EP3863722A2 - Variants d'anticorps anti-lap et leurs utilisations - Google Patents

Variants d'anticorps anti-lap et leurs utilisations

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Publication number
EP3863722A2
EP3863722A2 EP19797430.6A EP19797430A EP3863722A2 EP 3863722 A2 EP3863722 A2 EP 3863722A2 EP 19797430 A EP19797430 A EP 19797430A EP 3863722 A2 EP3863722 A2 EP 3863722A2
Authority
EP
European Patent Office
Prior art keywords
antibody
seq
lap
nos
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP19797430.6A
Other languages
German (de)
English (en)
Inventor
Randall Burton
Jessie M. ENGLISH
Barbara S. Fox
Stavros KOPSIAFTIS
Yacob Gomez Llorente
Renee MOORE
Patricia Rao
Giovanna Scapin
Kenneth J. Simon
Haihong Zhou
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tilos Theapeutics Inc
Merck Sharp and Dohme LLC
Original Assignee
Tilos Theapeutics Inc
Merck Sharp and Dohme LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tilos Theapeutics Inc, Merck Sharp and Dohme LLC filed Critical Tilos Theapeutics Inc
Publication of EP3863722A2 publication Critical patent/EP3863722A2/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to anti-LAP antibodies or antigen binding fragments thereof. Another aspect of the invention relates to compositions and kits comprising the anti-LAP antibodies or antigen binding fragments. Another aspect of the invention relates to methods for treating diseases, for example cancer, by administering the antibodies or antigen binding fragments.
  • Transforming growth factor beta 1 (TGFpi ) is synthesized as a pro-protein complex, in which the mature cytokine is caged within LAP (latency associated peptide), which is the latency associated peptide of TGFP 1.
  • LAP latency associated peptide
  • the LAP-TGFpi complex is disulfide bonded to one of five currently known anchor proteins: Glycoprotein A repetitions predominant (GARP), Leucine -rich repeat-containing protein 33 (LRRC33), Latent-transforming growth factor beta-binding protein 1 (LTBP1), Latent-transforming growth factor beta-binding protein 3 (LTBP3), and Latent- transforming growth factor beta-binding protein 4 (LTBP4).
  • GRP Glycoprotein A repetitions predominant
  • LRRC33 Leucine -rich repeat-containing protein 33
  • LTBP1 Latent-transforming growth factor beta-binding protein 1
  • LTBP3 Latent-transforming growth factor beta-binding protein 3
  • LTBP4 Latent-
  • GARP also referred to as leucine-rich repeat protein 32 or LRRC32
  • LRRC32 is a transmembrane protein that anchors LAP-TGFpi to the surface of lymphocytes, most notably regulatory T cells. GARP is also expressed on platelets, B cells, NK cells, fibroblasts, mesenchymal stromal cells, mesenchymal stem cells, and endothelial cells and also governs LAP-TGFP 1 expression on those cell types.
  • LRRC33 is a transmembrane protein that is reported to anchor LAP-TGFP 1 to the surface of myeloid cells, most notably macrophages, dendritic cells, and myeloid derived suppressor cells (MDSCs).
  • LTBP1, LTBP3, and LTBP4 are secreted molecules that anchor LAP-TGFp 1 into the extracellular matrix (ECM).
  • LAP binding agents have been used in the art as tools to identify certain cell populations, little is known about LAP’s relevance in disease states.
  • the location of the LAP-TGFpi complex is of critical biological and clinical importance because, once the mature TGFP 1 cytokine, which has a short half-life in solution, is released, it acts locally, either in an autocrine or near paracrine fashion. Therefore, the anchor proteins are a principal mechanism whereby latent TGFpi is staged in a specific location, awaiting the release of the potent mature cytokine to act on the local tissue.
  • LAP-TGFp 1 has different functions when expressed in different locations.
  • LAP-TGFp 1 anchored by LTBPs in the extracellular matrix is of primary importance for tissue homeostasis.
  • Xu et al. ⁇ Bone Research 20l8;6:2) noted that“the TGF-b complex is more like a molecular sensor that responds instantly to ECM perturbations through the release of an active ligand that exerts physiological effects at a cellular level, thus ensuring normal tissue homeostasis.”
  • LAP-TGFpi incorporation into the extracellular matrix are known to result in human disease. For example, deletion of LTBP-3 in both mice and humans results in similar defects in both bone and dental formation. LTBP-3 defects are also associated with the aortic dilation seen in Marfan syndrome (Rifkin et al., Matrix Biol 20l8;7l-72:90-99). These effects are believed to be due to aberrant direct effects of TGFP 1 in the local extracellular matrix (Xu et al, Bone Research 20l8;6:2).
  • LAP- TGFp 1 anchored by GARP is of primary importance for the immunosuppressive function of regulatory T cells (Edwards et al, Eur J Immunol 2016;46:1480-9) and of suppressive B cell subpopulations (Wallace et al, JCI Insight 20l8;3:e99863).
  • Some tumors have also been shown to express GARP, allowing them to locally express TGFP and directly suppress the immune system in the tumor microenvironment and support their own growth (Metelli et al, Journal of Hematology & Oncology 20l8;l l:24).
  • LAP-TGFp 1 anchored to myeloid cells is of primary importance for the
  • An aspect of the invention provided herein is a construct (e.g., polynucleotide, expression vector and host cell), protein or peptide comprising any of the sequences described herein, for example, the amino acid sequences found in tables such as Table 34.
  • a construct e.g., polynucleotide, expression vector and host cell
  • protein or peptide comprising any of the sequences described herein, for example, the amino acid sequences found in tables such as Table 34.
  • antibodies and antigen binding fragments thereof that bind LAP comprising the structural and functional features specified below (e.g., any one of the amino acid sequences of SEQ ID NOs: 16-197, 214, 216-240, 242-245, 248, 249 and 255 in Table 34).
  • the antibodies and antigen binding fragments comprise the amino acid sequences described in the tables herein, e.g., SEQ ID NOs: 16-197, 214, and 216-255.
  • the LAP comprises a complex and/or an epitope comprising LAP and a TGFP (e.g., T ⁇ Eb 1 ).
  • TGFP e.g., T ⁇ Eb 1
  • the epitope is described in examples herein, e.g., Examples 19-23.
  • An aspect of the invention provides isolated monoclonal antibodies (e.g., recombinant humanized, chimeric, and human antibodies) which exhibit therapeutically advantageous patterns of binding to LAP-TGEP 1 (e.g., human LAP-TGEP 1 ) and functional properties compared to prior anti-LAP antibodies.
  • the anti-LAP antibodies selectively bind to LAP-TGFp 1 on cells (e.g., immune cells and other immunosuppressive cells) but not to LAP-TGFp 1 in the extracellular matrix, and thus are able to target a broad range of clinically relevant cell types while sparing the natural function/activation of LAP-TGFpl in the
  • the antibodies described herein provide the clinical benefit of inhibiting TGFP activation and release of the mature cytokine in a highly selective, cell-specific manner.
  • the anti-LAP antibodies are of an isotype with active effector function and enhanced binding of a specific anti- LAP antibody to a given cell population will result in increased depletion of that cell population by ADCC or CDC. Accordingly, anti-LAP antibodies disclosed herein are ideal for treating a broad variety of diseases, including cancers and other diseases involving immunosuppressive cells, both in monotherapy and combination with other immunomodulatory or therapeutic agents (e.g., immune checkpoint inhibitors).
  • an antibody e.g., recombinant humanized, chimeric, domain, or human antibody
  • an antibody e.g., recombinant humanized, chimeric, domain, or human antibody
  • antigen binding fragment thereof which specifically binds to LAP comprising:
  • a heavy chain variable region comprising complementarity determining region (CDR) 1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 16, 26, and 18, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 19, 20, and 21, respectively;
  • CDR complementarity determining region
  • the antibody is a humanized antibody, chimeric antibody, or human antibody.
  • the administering step (e.g., in a method of treating or diagnosing a subject) is performed with the antibody.
  • the antibody is a humanized antibody, chimeric antibody or human antibody.
  • the LAP is human LAP, cynomolgus monkey (cyno) LAP, rat LAP, and/or mouse LAP.
  • the administering step (e.g., in a method of treating or diagnosing a subject) is performed with the antigen binding fragment.
  • the constant region of the antibody is a human IgGl constant region.
  • the IgGl constant region comprises the amino acid sequence set forth in a table disclosed herein, (e.g., Table 34).
  • the IgGl constant region comprises the amino acid sequence set forth in SEQ ID NOs: 196, 244, or 245.
  • the constant region of the antibody is a human IgG4 constant region.
  • the human IgG4 constant region comprises the amino acid sequence set forth in SEQ ID NO: 197.
  • an isolated antibody or antigen binding fragment which specifically binds to human LAP and comprises heavy and light chain variable region sequences which are at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 42 and 52, respectively; (b) SEQ ID NOs: 101 and 104, respectively; (c) SEQ ID NOs: 98 and 104, respectively; (d) SEQ ID NOs: 133 and 154, respectively; and (e) SEQ ID NOs: 218 and 154, respectively.
  • an isolated antibody or antigen binding fragment which specifically binds to human LAP and comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 218 or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 218 with 1, 2, or 3 amino acid substitutions; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 154 or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 154 with 1, 2, or 3 amino acid substitutions.
  • at least one substitution is located within a CDR.
  • at least one substitution is located within a framework region.
  • at least one substitution is located within at least one CDR and at least one the framework region.
  • any and/or ah of the at least one substitution is located and/or found within the framework region(s).
  • the antibody is a humanized antibody, chimeric antibody, or human antibody.
  • Another aspect of the invention provided herein is an isolated antibody or antigen binding fragment which specifically binds to human LAP and comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 218 or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 218 with 1-5, 5-10, 10-15, 15-20, or 20-25 amino acid substitutions; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 154 or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 154 with 1-5, 5-10, 10-15, 15-20, or 20-25 amino acid substitutions.
  • At least one substitution is located in at least one CDR region.
  • the at least one substitution is located within multiple CDRs.
  • the at least one substitution is located within at least one framework region.
  • the at least one substitution is located within at least one CDR and in at least one framework region.
  • any and/or ah of the at least one substitution is located and/or found within the framework region(s).
  • the antibody is a humanized antibody, chimeric antibody, or human antibody.
  • Another aspect of the invention provided herein is an isolated antibody or antigen binding fragment which specifically binds to human LAP, wherein the antibody or antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 218, wherein the antibody or antigen binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 154.
  • the antibody is a humanized antibody, chimeric antibody, or human antibody.
  • Another aspect of the invention provided herein is an isolated antibody or antigen binding fragment which specifically binds to human LAP, wherein the antibody or antigen binding fragment comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 218, wherein the antibody or antigen binding fragment comprises a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 154.
  • the antibody is a humanized antibody, chimeric antibody, or human antibody.
  • Another aspect of the invention provided herein is an isolated antibody or antigen binding fragment which specifically binds to human LAP, wherein the antibody or antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 219, wherein the antibody or antigen binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 155.
  • the antibody is a humanized antibody, chimeric antibody, or human antibody.
  • Another aspect of the invention provided herein is an isolated antibody or antigen binding fragment which specifically binds to human LAP, wherein the antibody or antigen binding fragment comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 219, wherein the antibody or antigen binding fragment comprises a light chain consisting of the amino acid sequence of SEQ ID NO: 155.
  • the antibody is a humanized antibody, chimeric antibody, or human antibody.
  • an isolated antibody or antigen binding fragment which specifically binds to human LAP and comprises heavy and light chain sequences which are at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 43 and 53, respectively; (b) SEQ ID NOs: 45 and 53, respectively; (c) SEQ ID NOs: 102 and 105, respectively; (d) SEQ ID NOs: 103 and 105, respectively; (e) SEQ ID NOs: 99 and 105, respectively; (f) SEQ ID NOs: 100 and 105, respectively; (g) SEQ ID NOs: 134 and 155, respectively; (h) SEQ ID NOs: 135 and 155, respectively; (i) SEQ ID NOs: 219 and 155, respectively; and (j) SEQ ID NOs: 220 and 155, respectively.
  • At least one substitution is located in at least one CDR region.
  • the at least one substitution is located within multiple CDRs.
  • the at least one substitution is located within at least one framework region.
  • the at least one substitution is located within at least one CDR and in at least one framework region.
  • any and/or all of the at least one substitution is located and/or found within the framework region(s).
  • the antibody is a humanized antibody, chimeric antibody, or human antibody.
  • an isolated antibody or antigen binding fragment which binds to human LAP and comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 110, 120, and 112, respectively, and a light chain variable region comprising CDR1,
  • the antibody comprises a human IgGl constant region.
  • the antibody is a humanized antibody, chimeric antibody, or human antibody.
  • an isolated antibody or antigen binding fragment which binds to human LAP and comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising amino acid sequences that are at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequences SEQ ID NOs: 110, 120, and 112, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising amino acid sequences that are at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequences of SEQ ID NOs: 113, 114, and 115, respectively, wherein the antibody further comprises a human IgGl constant region.
  • the antibody is a humanized antibody, chimeric antibody, or human antibody.
  • an isolated antibody or antigen binding fragment which binds to human LAP and comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 110, 120, and 112, respectively, and a light chain variable region comprising CDR1,
  • the antibody is a humanized antibody, chimeric antibody, or human antibody.
  • An aspect of the invention provides an anti-LAP antibody or antigen-binding fragment thereof described herein (e.g., 20E6 and humanized versions thereof described in Table 34) is in association with an isolated antibody comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 240 and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 241.
  • An aspect of the invention provides anti-LAP antibody or antigen-binding fragment thereof described herein (e.g., 20E6 and humanized versions thereof described in Table 34) is in association with an isolated antibody comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 246 and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 247.
  • an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 246
  • an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 247.
  • an isolated antibody or antigen binding fragment which binds to the same epitope on LAP as the anti-LAP antibodies or antigen binding fragments described herein.
  • an isolated antibody or antigen binding fragment which binds to the same amino acids or groups of amino acids on LAP as the anti-LAP antibodies or antigen binding fragments described herein.
  • the epitope e.g., LAP and a LAP complex comprising LAP and TBRbI
  • antibody, or antigen binding fragment has the characteristics described herein, such as Tables 25, 26, 27, 28, 29, and/or 30.
  • the anti-LAP antibody binds to specific amino acids of human LAP, for example amino acids 31-40, 274-280, and 340-343 of human LAP-TGPpl (SEQ ID NO: 1), e.g., as assessed by at least one structural analytical method such as crystallography and/or cryo-EM.
  • the anti-LAP antigen binding fragment binds to specific amino acids of human LAP, for example amino acids 31-40, 274-280, and 340-343 of human LAP-TGPpl (SEQ ID NO: 1), e.g., as assessed by at least one structural analytical method such as crystallography and/or cryo-EM.
  • the antibody or the antigen binding fragment binds to one or more amino acids within the recited amino acids, i.e., one or more amino acids within amino acids 31-40, 274-280, and 340-343 of human LAP-TGFP 1 (SEQ ID NO: 1).
  • the isolated antibody or antigen binding fragment binds to one or more residues of residues 31-40, 274-280, and 340-343 of human LAP-TGFpl (SEQ ID NO: 1), or binds to one or more residues of residues 31-43, 272-283, and 340-344 of human LAP-TGFpl (SEQ ID NO: 1).
  • the anti-LAP antibody or antigen binding fragment thereof binds to a specific region or regions of LAP-TGPpl, for example, Region 1, Region 2, Region 3, and/or Region 4 as shown in Ligure 34, e.g., as assessed by at least one structural analytical method such as HDX-MS.
  • the antibody or antigen binding fragment binds to human LAP (e.g., with a K D of about 11 nm, with a K D of 11 nM or less, or with a K D of 10 nM or less). In various embodiments, the antibody or antigen binding fragment binds to LAP (e.g., human, cyno, rat or mouse) with a K D of 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, or 10 nM or less.
  • LAP e.g., human, cyno, rat or mouse
  • the antibody or antigen binding fragment binds to human LAP (e.g., with a K D of less than 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, or 10 nM). In some embodiments, the antibody or antigen binding fragment binds to human LAP with a KD of about 40-60 nM, or about 50-60 nM. In various embodiments, the KD IS determined by Octet binding analysis. In various embodiments, the KD is determined by BIACORE® surface plasmon resonance (referred to interchangeably as“BiaCore” and“BIACore”) binding analysis.
  • the antibody or antigen binding fragment has a binding affinity described herein, e.g., Tables 31-32.
  • the antibody or antigen binding fragment inhibits TGFP 1 activation.
  • the antibody or antigen binding fragment inhibits integrin activation of TGFP and/or release of human LAP from the LAP-TGFp 1 complex.
  • the antibody or antigen binding fragment binds to both human and murine LAP.
  • the antibody or antigen binding fragment binds to human LAP in the absence of an anchor protein.
  • the antibody or antigen binding fragment binds or is determined to bind to LAP-TGFpi complexed with an anchor protein (e.g., GARP, LRRC33) on immunosuppressive cells, but does not bind to the anchor protein or to an epitope composed of residues of both LAP-TGFP and the anchor protein.
  • Immunosuppressive cells include, for example, suppressive T cells (e.g., regulatory T cells, activated T cells), cancer-associated fibroblasts, M2 macrophages, cancer cells expressing LAP- TGFpl, and/or monocytic myeloid-derived suppressor cells.
  • the antibody or antigen binding fragment does not bind free TGFpi or empty LAP.
  • the antibody or antigen binding fragment does not bind to LAP in extracellular matrix. In some embodiments, the antibody or antigen binding fragment does not bind or is determined to not to bind to LAP complexed with LTBP1, LTBP3 and/or LTBP4. In some embodiments, the antibody or antigen binding fragment binds or is determined to bind to human LAP-TGFP 1 comprising K27C and Y75C mutations and/or all or a portion of (e.g., within) residues 82-130 of human LAP-TGFpl (SEQ ID NO: 1), but not human LAP-TGFpl comprising the Y74T mutation.
  • the antibody or antigen binding fragment binds or is determined to bind to both GARP-positive immunosuppressive cells and GARP-negative immunosuppressive cells. In some embodiments, the antibody or antigen binding fragment binds or is determined to bind to platelets, but does not cause platelet aggregation or platelet degranulation. In some embodiments, the antibody is an IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody, or variant thereof. In some embodiments, the antibody is a chimeric, domain, humanized, or human antibody.
  • the antibody or antigen binding fragment thereof can comprise any of the variable light chains described herein and light chain constant domain described herein (e.g ., a human light chain constant domain).
  • the light chain constant domain is recited in Table 34.
  • the antibody or antigen binding fragment thereof comprises a human kappa light chain constant domain or a variant thereof.
  • the variant comprises up to 1-25 modified amino acid substitutions (e.g., 20 substitutions).
  • the antibody or antigen binding fragment thereof comprises a human lambda light chain constant domain or a variant thereof.
  • the variant comprises up to 1-25 modified amino acid substitutions (e.g., 20 substitutions).
  • the antibody or antigen binding fragment thereof comprises a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 256.
  • a bispecific antibody comprising a first binding region with a specificity for LAP of an anti-LAP antibody described herein, and a second binding region or therapeutic agent which binds to another antigen, e.g., a tumor- associated antigen, CD4, CD8, CD45, CD56, CD14, CD16, CD19, CDl lb, CD25, CD20, CD22, CD30, CD38, CD114, CD23, CD73, CD163, CD206, CD203, CD200R or CD39.
  • the second binding region or therapeutic agent which binds to a receptor protein.
  • an immunoconjugate comprising an anti-LAP antibody or antigen binding fragment described herein linked to a detectable moiety, a binding moiety, a labeling moiety, and/or a biologically active moiety, e.g., a bispecific molecule and/or a bifunctional molecule.
  • the biologically active moiety comprises a receptor trap construct.
  • nucleic acid comprising a nucleotide sequence that encodes the heavy and/or light chain variable region of an anti-LAP antibody or antigen binding fragment described herein, as well as expression vector(s) comprising the same, and cells transformed with the expression vector(s).
  • nucleic acid comprising a nucleotide sequence that encodes the heavy chain and/or light chain of an anti-LAP antibody or antigen binding fragment described herein, as well as expression vector(s) comprising the same, and cells transformed with the expression vector(s).
  • a pharmaceutical composition comprising an anti-LAP antibody or antigen binding fragment described herein and a pharmaceutically acceptable carrier.
  • the composition comprises one or more additional therapeutic agents, such as an anti-cancer agent, a chemotherapeutic agent, an immunomodulatory agent (e.g., an immunostimulatory agent or immunosuppressive agent), an anti-inflammatory agent, and/or an immune checkpoint blocker (e.g., an anti-PD-l antibody, an anti-PD-Ll antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody, and an anti-TIM3 antibody).
  • the PD-l antibody is pembrolizumab.
  • an anti-LAP antibody or antigen-binding fragment thereof described herein is in association with an isolated antibody comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 240 and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 241.
  • SEQ ID NOs: 240 and 241 correspond to the heavy chain and light chain sequences of pembrolizumab.
  • an anti-LAP antibody or antigen-binding fragment thereof described herein is in association with an isolated antibody comprising an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 246 and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 247.
  • SEQ ID NOs: 246 and 247 correspond to the heavy and light chain sequences of pembrolizumab.
  • kits comprising an anti-LAP antibody or antigen binding fragment described herein and instructions for use.
  • an antibody that specifically binds to LAP comprising: (a) immunizing an animal with a polypeptide comprising an epitope on human LAP recognized by 28G11, (b) selecting from the immunized animal an antibody that binds to the same epitope as 28G11, and (c) isolating the antibody selected from step (b).
  • the antibody binds to a human LAP complex comprising TGFpi .
  • the antibody binds to all or a portion (e.g., within) residues 82-130 of human LAP.
  • TGFp 1 activation on cells e.g., immunosuppressive cells such as suppressive T cells (e.g., regulatory T cells, activated T cells), M2 macrophages, cancer cells expressing LAP-TGFpl, cancer-associated fibroblasts, mesenchymal stromal cells mesenchymal stem cells, and/or monocytic myeloid-derived suppressor cells
  • immunosuppressive cells such as suppressive T cells (e.g., regulatory T cells, activated T cells), M2 macrophages, cancer cells expressing LAP-TGFpl, cancer-associated fibroblasts, mesenchymal stromal cells mesenchymal stem cells, and/or monocytic myeloid-derived suppressor cells
  • TGFpi activation on extracellular matrix comprising administering to the subject any anti-LAP antibody or antigen binding fragment, bispecific molecule, immunoconjugate, and/or pharmaceutical composition described herein.
  • provided herein is a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of any anti-LAP antibody or antigen binding fragment, bispecific molecule, immunoconjugate, and/or pharmaceutical composition described herein.
  • the cancer is characterized by abnormal TGFP activity.
  • the cancer is associated with infiltration of cluster of differentiation 4 (CD4)+ regulatory T cells, cluster of differentiation 8 (CD8)+ regulatory T cells, regulatory B cells, myeloid-derived suppressor cells, tumor-associated macrophages, cancer-associated fibroblasts, and/or innate lymphoid cells.
  • the cancer is breast cancer, bladder cancer, uterine/cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, neoplasm of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, or myelodysplastic syndromes.
  • one or more additional therapies is administered, for example, radiation therapy, chemotherapy, an immune checkpoint inhibitor (e.g., an anti-PD-l antibody, an anti-PD-Ll antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody, and an anti-TIM3 antibody), immunostimulatory therapy, immunosuppressive therapy, cell therapy, and a therapeutic agent (e.g., anti-cancer agent, a chemotherapeutic agent, an immunosuppressive agent, an immunomodulatory agent, and an anti inflammatory agent).
  • an immune checkpoint inhibitor e.g., an anti-PD-l antibody, an anti-PD-Ll antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody, and an anti-TIM3 antibody
  • immunostimulatory therapy e.g., an anti-cancer agent, a chemotherapeutic agent, an immunosuppressive agent, an immunomodulatory agent, and an anti inflammatory agent.
  • a method of detecting LAP comprising contacting a sample (e.g ., a biological sample) with any anti-LAP antibody or antigen binding fragment, bispecific molecule, immunoconjugate, and/or pharmaceutical composition described herein, and detecting the complex.
  • a method of diagnosing a cancer associated with regulatory T cell infiltration comprising contacting a biological sample from a patient afflicted with the cancer with any anti-LAP antibody or antigen binding fragment, bispecific molecule, immunoconjugate, and/or pharmaceutical composition described herein, wherein positive staining with the antibody or antigen binding fragment, bispecific molecule, immunoconjugate, and/or pharmaceutical composition indicates the cancer is associated with regulatory T cell infiltration.
  • a method of diagnosing a cancer associated with GARP-negative suppressive cells comprising contacting a biological sample from a patient afflicted with the cancer with any anti-LAP antibody or antigen binding fragment, bispecific molecule, immunoconjugate, and/or pharmaceutical composition described herein which binds to GARP-negative suppressive cells, wherein positive staining with the antibody or antigen binding fragment, bispecific molecule, immunoconjugate, and/or pharmaceutical composition and negative staining with an anti-GARP antibody indicates the cancer is associated with GARP-negative suppressive cells.
  • provided herein is a method of selecting a patient afflicted with cancer for treatment with an anti-LAP antibody or antigen binding fragment, bispecific molecule, immunoconjugate, and/or pharmaceutical composition described herein comprising contacting a biological sample from the patient with the antibody or antigen binding fragment, bispecific molecule, immunoconjugate, and/or pharmaceutical composition, wherein positive staining with the antibody or antigen binding fragment indicates the cancer is amenable to treatment with the antibody.
  • a method of determining the response of a patient afflicted with cancer to treatment with an anti-LAP antibody or antigen binding fragment described herein comprising contacting a biological sample from the patient with the antibody or antigen binding fragment, wherein reduced staining with the antibody or antigen binding fragment, bispecific molecule, immunoconjugate, and/or pharmaceutical composition indicates the cancer is responding to treatment with the antibody.
  • a method of making an antibody that specifically binds to the same epitope on human LAP recognized by 28G11 comprising immunizing an animal with an immunogen comprising a peptide, wherein the peptide comprises the epitope recognized by 28G11, selecting from the immunized animal an antibody that binds to the same epitope as 28G11, and obtaining an antibody that binds to the same epitope as 28G11.
  • the human LAP comprises a complex comprising human LAP and TGFpl.
  • Another aspect of the invention are uses of any of the anti-LAP antibodies or antigen binding fragments, bispecific molecules, immunoconjugates, and/or pharmaceutical
  • immunosuppressive cells but not TGFp 1 activation on extracellular matrix; treating cancer; diagnosing a cancer (e.g ., a cancer associated with regulatory T cell infiltration or GARP- negative suppressive cells); selecting a patient afflicted with cancer; and determining the response of a patient afflicted with cancer to treatment with the anti-LAP antibodies described herein.
  • a cancer e.g ., a cancer associated with regulatory T cell infiltration or GARP- negative suppressive cells
  • selecting a patient afflicted with cancer e.g a cancer associated with regulatory T cell infiltration or GARP- negative suppressive cells
  • selecting a patient afflicted with cancer e.g a cancer associated with regulatory T cell infiltration or GARP- negative suppressive cells
  • selecting a patient afflicted with cancer e.g a cancer associated with regulatory T cell infiltration or GARP- negative suppressive cells
  • selecting a patient afflicted with cancer e.g a cancer associated with regulatory T cell infiltration or GARP- negative
  • Figures 1A-1F are graphs showing the binding of antibodies 28Gl l_hIgGl,
  • Figures 2A-2F are graphs showing the binding of antibodies 28Gl l_MgGl,
  • HTl080-huBl HT1080 cells overexpressing LAP-TGEP 1
  • HTl080-K27C_Y75C HT1080 cells overexpressing LAP-TGFpl with K27C and Y75C mutations (mutations that prevent T ⁇ Eb 1 activation by integrins;“closed” conformation”)
  • HT1080-Y74T HT1080 cells overexpressing LAP-TGEP 1 with a Y74T mutation (mutation that favors spontaneous release of TGFpl (“open conformation”)
  • HTl080-ch2.3 HT1080 cells overexpressing chimeric LAP-TGEpi in which exon 2.3 (residues 131-164) of human LAP- TGFp 1 have been replaced with corresponding residues from chicken LAP-TGEP 1 (UniProt accession #H9CX0l);
  • HTl080-ch4 HT1080 cells overexpressing chimeric LAP-TGEP 1 in which exon 4 (residue
  • Figures 3A-3F are graphs showing the binding of antibodies 28Gl l_MgGl,
  • HTl080-huBl HT1080 cells overexpressing LAP-TGEP 1
  • HTl080-emptyLAP HT1080 cells overexpressing LAP which does not include the mature TGFp 1 cytokine
  • HTl080-ch2.2 HT1080 cells overexpressing chimeric LAP-TGEP 1 in which exon 2.2 (residues 108-130) of human LAP-TGFpl has been replaced with exon 2.2 from chicken LAP-TGFpl.
  • Figure 4 is a graph showing the binding of antibodies 28Gl l_hIgGl, 22F9_hIgGl, 20E6_hIgGl, l7G8_MgGl, 24E3_MgGl, and 1D11 to mature TGFp (i.e., TGFp without LAP), as measured by an ELISA assay in which inhibition of signal reflects binding to mature TGFp.
  • Figures 5A and 5B are sets of graphs showing the effects of 28G11 on TGEpi activation in P3U1 cells expressing human LAP-TGEP 1 ( Figure 5A) and murine LAP-TGEpi ( Figure 5B).
  • Figures 6A-6F are graphs showing the effects of antibodies ) 17G8 (Figure 6A for human LAP-TGEpi and Figure 6B for mouse LAP-TGFpl), 24E3 ( Figure 6C for human LAP- TGF l and Figure 6D for murine LAP-TGP ’ P 1 ), 22F9 ( Figure 6E for human LAP-TGF l), 20E6
  • Figure 6F for human LAP-TGFpi on TGEpi activation in P3U1 cells expressing human or mouse LAP-TGFp 1.
  • Figures 6G-6L are graphs showing the effects of 28Gl l_IgG2a (Figure 6G), 20E6_IgG2a (Figure 6H), 22F9_IgG2a ( Figure 61), 24E3_MgGl ( Figure 6J), l7G8_MgGl ( Figure 6K), and 20E6_H0.2aLl_hIgGl ( Figure 6L) on TGFpi activation in R3EG1 cells expressing human or mouse LAP-TGFpi .
  • Figure 7 is a graph showing binding of the indicated anti-LAP antibodies to extracellular matrix (ECM) deposited by R3EG1 cells.
  • ECM extracellular matrix
  • Three cell types were tested: R3EG1 cells without LAP- TGFp, R3EG1 cells expressing human LAP-TGFpi , and R3EG1 cells expressing murine LAP- TGFp 1.
  • Antibodies were used at a concentration of 2 pg/mL.
  • Figure 8 is a graph showing the dose-response relationship in binding of the indicated anti-LAP antibodies (i.e., antibodies 28G11, 22F9, 20E6, 17G8, and 24E3) to human platelets.
  • the indicated anti-LAP antibodies i.e., antibodies 28G11, 22F9, 20E6, 17G8, and 24E3
  • Figures 9A-9E are graphs showing the effects of anti-LAP antibodies 28Gl l_hyb
  • Figures 10A and 10B are graphs showing the binding of the indicated anti-LAP antibodies to THP-l cells. THP cell binding is expressed as the percentage (%) of LAP+ cells in Figure 10A and as median fluorescent intensity (MFI) in Figure 10B.
  • Figure 10C is a graph showing the binding of the indicated anti-LAP antibodies to THP-l cells. THP cell binding is expressed as the % of LAP+ cells.
  • Figure 10D shows dot plots for the binding of the indicated anti-LAP antibodies to THP-l cells.
  • Figures 10E and 10F are graphs showing the binding of the indicated anti-LAP antibodies to U937 cells. U937 cell binding is expressed as the % of LAP+ cells in Figure 10E and as MFI in Figure 10F.
  • Figure 10G shows dot plots for the binding of the indicated anti-LAP antibodies to U937 cells.
  • Figure 11 is a graph showing the binding of anti-LAP antibodies 28G11, 22F9, and 20E6 to regulatory T cells, CD8 cells, M2 macrophages, and M-MDSCs from CT26 TILs, expressed as the percentage of positive cells.
  • Figure 12 is a graph showing the ability of humanized 28G11 variants to compete with murine 28G11 parental antibody for binding to P3U1 cells over-expressing human GARP and LAP-TGFp 1.
  • H0-H2 are variant humanized heavy chains
  • L0-L3 are variant humanized light chains.
  • Figure 13 is a graph showing the binding of the indicated humanized 28G11 variants to P3U1 cells over-expressing human GARP and LAP-TGFP 1.
  • Figures 14A-14D are graphs showing the inhibition of TGEpi activation by the indicated humanized 28G11 variants by ELISA.
  • Figure 15A is a graph showing the effects that various combinations of reversions in the humanized 28G1 l_H2L3 variant back to human residues have on binding to HT1080 cells overexpressing human LAP-TGEP 1.
  • the various antibodies tested are listed as HC variant_LC variant.
  • Figure 15B is a graph showing the effects that the 28Gl l_H2.lL3 and 28Gl l_H2aL3a variants have on binding to HT1080 cells overexpressing human LAP-TGEpi .
  • Black bars correspond to antibody binding to HT1080 cells over-expressing human LAP-TGEP 1.
  • Gray bars correspond to antibody binding to control HT1080 cells which do not over-express human LAP- TGFpl.
  • Figure 16 is a graph showing the binding of the indicated humanized 22F9 variants to HT1080 cells over-expressing human LAP-TGEP 1. Black bars correspond to antibody binding to HT1080 cells over-expressing human LAP-TGEpi . Gray bars correspond to antibody binding to control HT1080 cells which do not over-express human LAP-TGEP 1.
  • Figures 17A and 17B are chromatograms from size exclusion high-performance liquid chromatography (SE-HPLC) for the indicated antibody 22F9 and variants.
  • Figure 18A is a graph showing the effects of reverting murine residues in the heavy chain of 22F9_H5L0 back to corresponding human residues on binding to HT1080 cells over expressing human LAP-TGEP 1.
  • Figure 18B is a graph showing the effects of substitutions to remove potential deamidation and/or isomerization sites in 22F9_H5L0 on binding to HT1080 cells over-expressing human LAP-TGEpi .
  • 22F9_H5.2L0 corresponds to 22F9_H5L0 with the double N54Q/D102A mutation.
  • Figure 18C is a graph showing the binding of 22F9_H0.l, 22F9_Hl.l, 22F9_H2.l, and 22F9_H3.l variants to HT1080 cells over-expressing human LAP- TGFp 1. Black bars correspond to antibody binding to HT1080 cells over-expressing human LAP-TGFp 1. Gray bars correspond to antibody binding to control HT1080 cells which do not over-express human LAP-TGFpi .
  • Figures 19A and 19B are graphs showing the binding of the indicated humanized 20E6 variants to human LAP-TGFp 1. Black bars correspond to antibody binding to HT1080 cells over-expressing human LAP-TGFpi . Gray bars correspond to antibody binding to control HT1080 cells which do not over-express human LAP-TGFpi .
  • Figure 20A is a graph showing the effects of murine back-substitutions to the Ll light chain of 20E6 on binding to human LAP-TGFP 1.
  • Figure 20B is a graph showing the binding of the 20E6_H0 heavy chain variant with reduced immunogenicity on binding to human LAP- TGFp 1. Black bars correspond to antibody binding to HT1080 cells over-expressing human LAP-TGFp 1. Gray bars correspond to antibody binding to control HT1080 cells which do not over-express human LAP-TGFpi .
  • Figures 21A-21C are bio-layer interferometry curves showing the binding of
  • 20E6_H0.2aLl_hIgGl antibody to Fc fusions comprising either human LAP-TGFpl, LAP- TGFp2, or LAP-TGFp3.
  • Figures 22A-22D are graphs showing the binding of multiple heavy and light chain CDR variants of 20E6_H0.2aLl_hIgGl antibody to human LAP-TGFpi .
  • Figures 23A and 23B are graphs showing the binding of F(ab’)2 fragments and Fab’ fragments of 20E6_H0.2aFl_hIgGl to P3FT1 cells overexpressing GARP and LAP-TGFP 1.
  • Figure 24 is a series of bio-layer interferometry curves showing the binding of MHG8 (a GARP-specific murine IgG2a) antibody, 16F4 (an anti-FAP) antibody, and humanized 20E6 antibody to soluble LAP-TGFpl, sGARP-LAP-TGFpl, and ECR3E-LAP-TGFpl complexes.
  • MHG8 a GARP-specific murine IgG2a
  • 16F4 an anti-FAP
  • humanized 20E6 antibody to soluble LAP-TGFpl
  • sGARP-LAP-TGFpl sGARP-LAP-TGFpl
  • ECR3E-LAP-TGFpl complexes ECR3E-LAP-TGFpl complexes.
  • Figure 25 is a schematic of the competition experiment used to compare binding epitopes of murine 28G11, 16F4, and MHG8, as described in Example 17.
  • Figure 26A shows an example of a cryo-EM map used to build the humanized 20E6- Fab/LAP-TGFp 1 model.
  • Figure 26B is a plot of the FSC curve versus resolution.
  • Figure 26C is the B-factor estimation using a Guinier plot.
  • Figure 27A is a cryo-EM map of the humanized 20E6 antibody complexed with LAP- TGFp 1 at 3.1 A resolution.
  • Figure 27B shows the electron density for PDB entry 5jxe at 2.9 A resolution.
  • Figure 28A shows interactions between LAP and the VL and VH of the humanized 20E6 Fab.
  • Figure 28B shows interactions between TGFP 1 and the humanized 20E6 VL and VH portions of the Fab.
  • Figure 28C and Figure 28D show additional interactions between LAP- TGFp 1 and the 20E6 VL portion of the 20E6 Fab. Constructs and interactions are shown in grayscale.
  • Figure 29A shows interactions between LAP and the VL and VH of the 28G11 humanized Fab.
  • Figure 29B shows interactions between TGFpi and the humanized 28G11 VL and VH portions of the Fab.
  • Figure 29C shows the repositioning of CDRs in humanized 28G11 and humanized 20E6.
  • Figure 29D shows that Q56 in humanized 28G11 is ideally positions to interact with the TGEpi loop spanning residues V342-K344.
  • Figure 29E shows that, in humanized 28G11, a Tyr residue is replaced by Gly, resulting in loss of interactions with the side chain of V341. Constructs and interactions are shown in grayscale.
  • FIG 30A shows the 2Mab:2TGFpl complex identified in cryo-EM (Mab refers to 22F9 Fab). Constructs are: LAP-TGEP 1 dimer; 22F9-Fabs; 22F9-Fcs.
  • Figure 30B provides examples of the 2D classes. Constructs and interactions are shown in grayscale.
  • Figure 31A shows interactions between LAP and the VL and VH portions of the 22F9 Fab.
  • Figure 31B shows interactions between TGFP and 22F9 VL and VH .
  • Figure 31C shows an overlay of 22F9-Fab + LAP-TGEP 1 complex and humanized 20E6-Fab + LAP-TGEpi (Fabs) and the different orientation of humanized 20E6-Fabs and 22F9-Fabs with respect to the antigen.
  • Figure 31D and Figure 31E show additional interactions between LAP-TGEP 1 and portions of the 20E6 Fab. Constructs and interactions are shown in grayscale.
  • Figure 32 is a H/D Difference Plot showing deuterium uptake protection upon
  • Figure 33 is a H/D Difference Plot showing deuterium uptake protection upon antibody binding to human LAP-TGEpi protein.
  • Figure 34 is a sequence alignment between human LAP-TGEP 1 and mouse LAP-TGEpi . Epitopes identified are all in regions that are homologous between human and mouse LAP- TGFp 1.
  • the human LAP-TGEpi sequence in the figure corresponds to SEQ ID NO: 257.
  • the mouse LAP-TGFp 1 sequence in the figure corresponds to SEQ ID NO: 7.
  • Figures 35A and 35B are size-exclusion chromatograms for human LAP-TGEpi , GARP- LAP-TGFp 1 , humanized 20E6, and complexes. The elution time for each sample is consistent with the gel filtration standard. Molecular weight reported was determined from the light scattering detector.
  • Figures 36A-36F are a series of graphs that show the effects of anti-LAP antibodies 28G11 and 16B4 in combination with an anti-PD-l antibody on tumor volume in a syngeneic CT26 colorectal cancer tumor model.
  • the data shown in the figures are: Figure 36A (anti-PD-l antibody alone), Figure 36B (28Gl l_IgG2a + anti-PD-l antibody), Figure 36C (IgG2a isotype control), Figure 36D (anti-PD-l antibody alone), Figure 36E (!6B4_IgG2a alone), and Figure 36F (l6B4_IgG2a + anti-PD-l antibody).
  • the anti-PD-l antibody was a rat anti-PD-l (clone RMPl-l4)-IgG2a antibody.
  • Figure 37 is a graph that shows the effects of anti-LAP antibody 28G11 in combination with an anti-PD-l antibody on tumor volume in a syngeneic EMT6 breast cancer tumor model.
  • the anti-PD-l antibody was a rat anti-PD-l (clone RMPl-l4)-IgG2a antibody.
  • the statistical test used was two-way ANOVA.
  • Figure 38 is a graph that shows the effects of anti-LAP antibody 22F9 in combination with an anti-PD-l antibody on tumor volume in a syngeneic EMT6 breast cancer tumor model.
  • the anti-PD-l antibody was a rat anti-PD-l (clone RMPl-l4)-IgG2a antibody.
  • the statistical test used was two-way ANOVA.
  • Figure 39 is a graph that shows the effects of anti-LAP antibody 20E6 in combination with an anti-PD-l antibody on tumor volume in a syngeneic EMT6 breast cancer tumor model.
  • the anti-PD-l antibody was a rat anti-PD-l (clone RMPl-l4)-IgG2a antibody.
  • the statistical test used was two-way ANOVA.
  • Figure 40 is a graph that shows the effects of anti-LAP antibodies 28G1 l_IgG2a and l6B4_IgG2a, and the anti-TGFp antibody lDl l_IgGl, as monotherapy on lung nodule counts in the 4T1 breast cancer tumor metastasis model (p ⁇ 0.05, unpaired T test following removal of outliers).
  • Figures 41A and 41B are graphs that show the effects of anti-LAP antibody
  • Figures 42A-42C are graphs that show the effects of anti-LAP antibody 28Gl l_IgG2a on CD73 expression in M-MDSCs (Figure 42A), M2 macrophages (Figure 42B), and dendritic cells (Figure 42C), with or without 12 Gy or 20 Gy radiation.
  • Figures 43A-43H are a series of graphs that show effects of anti-LAP antibodies 28G11- mIgG2a and 20E6-mIgG2a alone and in combination with anti-PD-l antibody on tumor volume in a syngeneic EMT6 mouse breast cancer tumor model.
  • the data shown in the figures are:
  • Figure 43A is a graph summary of efficacy of 28G1 l-mIgG2a and Figure 43B is a graph summary of efficacy of 20E6-mIgG2a.
  • Figure 43C tumor volume data for control antibodies
  • Figure 43D antibody 20E6-mIgG2a
  • Figure 43E antibody 28Gl l-mIgG2a
  • Figure 43F anti- PD-l antibody
  • Figure 43G a combination of antibody 20E6-mIgG2a and anti-PD-l antibody
  • Figure 43H antibody 28Gl l-mIgG2a and anti-PD-l antibody
  • Figures 44A-44D are a series of Biacore graphs showing binding of anti-LAP F(ab’) to human LAP-TGFP isoforms 1, 2, and 3.
  • Figures 45A-45D are a series of Biacore graphs showing binding of anti-LAP F(ab’) to human, cynomolgus monkey, rat, and mouse LAP-TGEp i .
  • Figure 46 is a graph showing inhibition of integrin (avb6) activation of LAP-TGFbl using 20E6_mIgG2a antibody, isotype control antibody or anti- anb6 (10D5) antibody.
  • “Abnormal” in the context of the activity, level or expression of a molecule means that the activity, level or expression is outside of the normal activity, level or expression for that molecule. “Normal” in the context of activity, level or expression refers to the range of activity, level or expression of the protein found in a population of healthy, gender- and age-matched subjects. The minimal size of this healthy population may be determined using standard statistical measures, e.g., the practitioner could take into account the incidence of the disease in the general population and the level of statistical certainty desired in the results.
  • the normal range for activity, level or expression of a biomarker is determined from a population of subjects (e.g., at least five, ten or twenty subjects), more preferably from a population of at least forty or eighty subjects, and even more preferably from more than 100 subjects.
  • LAP ency associated peptide
  • “LAP-TGFp i” and“LAP/TGFp i” are used interchangeably herein to refer to the human TGEp i precursor peptide (which includes the T ⁇ Eb 1 cytokine) and includes the amino acid sequence of SEQ ID NO: 1 (Uniprot splP0H37ITGFB l_HUMAN with signal sequence removed).
  • LAP can also refer to the amino-terminal domains of the human TGFP2 precursor peptide (LAP region: SEQ ID NO: 4, LAP-TGFP2: SEQ ID NO: 3) and human TGFP3 precursor peptide (LAP region: SEQ ID NO: 6, LAP-TGFP2: SEQ ID NO: 5), as well as their counterparts from other species (e.g., mouse TGFpl precursor peptide (mouse LAP region: SEQ ID NO: 8; mouse LAP-TGFpl: SEQ ID NO: 7), mouse TGFP2 precursor peptide (mouse LAP region: SEQ ID NO: 10; mouse LAP-TGFP2: SEQ ID NO: 9), and mouse TGFP3 precursor peptide (mouse LAP region: SEQ ID NO: 12; mouse LAP-TGFP3: SEQ ID NO: 11)) and other naturally occurring allelic, splice variants, and processed forms thereof.
  • mouse TGFpl precursor peptide mouse LAP region:
  • LAP is synthesized as a complex with TGFp.
  • LAP in the absence of mature TGFP is referred to as“empty LAP.”
  • “empty LAP” as used herein refers to LAP originating from the N-terminal domain of human TGFP 1.
  • the anti-LAP antibodies described herein may also bind to residues of mature TGFP within the LAP-TGFP 1 complex.
  • the antibody at least binds to residues in the LAP portion of the complex.
  • free TGFp i refers to the mature TGFP 1 cytokine, i.e., TGFpi that is not complexed with LAP.
  • anchor protein refers to a protein that anchors LAP-TGFP to a cell surface or to the extracellular matrix.
  • exemplary anchor proteins include GARP, LRRC33, LTBP1, LTBP3, and LTBP4.
  • GARP and LRRC33 are proteins that anchor LAP-TGFP to the surface of cells
  • LTBP1, LTBP3, and LTBP4 are proteins that anchor LAP-TGFP to the extracellular matrix.
  • the term“antibody” as used herein includes whole antibodies and any antigen binding fragments ⁇ i.e.,“antigen-binding portions”) or single chains thereof.
  • An“antibody” refers, in one embodiment, to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • VH heavy chain variable region
  • the heavy chain constant region is comprised of three domains, CH1, CH2, and CH3.
  • each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g ., effector cells) and the first component (Clq) of the classical complement system.
  • isotype refers to the antibody class (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE antibody) that is encoded by the heavy chain constant region genes.
  • antibody class e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE antibody
  • Antibodies typically bind specifically to their cognate antigen with high affinity, reflected by a dissociation constant (K D ) of 10 5 to 10 11 M or less. Any K D greater than about 10 4 M is generally considered to indicate nonspecific binding.
  • K D dissociation constant
  • an antibody that "binds specifically" to an antigen refers to an antibody that binds to the antigen and substantially identical antigens with high affinity, which means having a K D of 10 7 M or less, preferably 10 8 M or less, even more preferably 5 x 10 9 M or less, and most preferably between 10 8 M and 10 10 M or less, but does not bind with high affinity to unrelated antigens.
  • antigen-binding portion of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human and/or mouse LAP). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • an antigen e.g., human and/or mouse LAP
  • binding fragments of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker.
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CH1 domains
  • a F(ab')2 fragment
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody.
  • Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.
  • Antibody fragments within the scope of the present invention also include F(ab’)2 fragments which may be produced by enzymatic cleavage of an IgG by, for example, pepsin.
  • Fab fragments may be produced by, for example, reduction of F(ab’)2 with dithiothreitol or mercaptoethylamine.
  • a Fab fragment is a VL-CL chain appended to a VH-CH1 chain by a disulfide bridge.
  • a F(ab’)2 fragment is two Fab fragments which, in turn, are appended by two disulfide bridges.
  • the Fab portion of an F(ab’)2 molecule includes a portion of the Fc region between which disulfide bridges are located.
  • acceptor human framework refers to a framework comprising the amino acid sequence of a light chain variable domain (V L ) framework or a heavy chain variable domain (V H ) framework derived from a human immunoglobulin framework or a human consensus framework.
  • An acceptor human framework“derived from” a human immunoglobulin framework or a human consensus framework may have the same amino acid sequence as the naturally-occurring human immunoglobulin framework or human consensus framework, or it may have amino acid sequence changes compared to wild-type naturally-occurring human immunoglobulin framework or human consensus framework. In some embodiments, the number of amino acid changes are 10, 9, 8, 7, 6, 5, 4, 3, or 2, or 1.
  • the V L acceptor human framework is identical in sequence to the V L human immunoglobulin framework sequence or human consensus framework sequence.
  • A“multispecific antibody” is an antibody (e.g., bispecific antibodies, tri-specific antibodies) that recognizes two or more different antigens or epitopes.
  • binding protein as used herein also refers to a non-naturally occurring (or recombinant) protein that specifically binds to at least one target antigen.
  • A“bispecific” or“bifunctional antibody” is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.
  • Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et ah, J. Immunol. 148, 1547-1553 (1992).
  • Bifunctional antibodies include, for example, heterodimeric antibody conjugates (e.g., two antibodies or antibody fragments joined together with each having different specificities), antibody/cell surface-binding molecule conjugates (e.g., an antibody conjugated to a non-antibody molecule such as a receptor), and hybrid antibodies (e.g., an antibody having binding sites for two different antigens).
  • heterodimeric antibody conjugates e.g., two antibodies or antibody fragments joined together with each having different specificities
  • antibody/cell surface-binding molecule conjugates e.g., an antibody conjugated to a non-antibody molecule such as a receptor
  • hybrid antibodies e.g., an antibody having binding sites for two different antigens.
  • recombinant antibody refers to antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for immunoglobulin genes (e.g., human immunoglobulin genes) or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library (e.g., containing human antibody sequences) using phage display, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences (e.g., human immunoglobulin genes) to other DNA sequences.
  • a host cell transformed to express the antibody e.g., from a transfectoma
  • combinatorial antibody library e.g., containing human antibody sequences
  • Such recombinant antibodies may have variable and constant regions derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies can be subjected to in vitro mutagenesis and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • a “human” antibody refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. Also encompassed are antibodies derived from human germline immunoglobulin sequences that include normal somatic hypermutations which alter the germline immunoglobulin sequences relative to the wild-type germline immunoglobulin sequences.
  • a “humanized” antibody refers to an antibody in which some, most or all of the amino acids outside the CDR domains of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulins. In one embodiment of a humanized form of an antibody, some, most or all of the amino acids outside the CDR domains have been replaced with amino acids from human immunoglobulins, whereas some, most or all amino acids within one or more CDR regions are unchanged. Any additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not abrogate the ability of the antibody to bind to a particular antigen.
  • a "humanized” antibody retains an antigenic specificity similar to that of the original antibody.
  • a “chimeric antibody” refers to an antibody in which the variable regions are derived from one or more species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody. See U.S. Pat. No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad Sci. USA 81: 6851-6855.
  • the term“monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies in the population are substantially similar and bind the same epitope(s) (e.g., the antibodies display a single binding specificity and affinity), except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts.
  • “Monoclonal” indicates the character of the antibody as having been obtained from a substantially homogenous population of antibodies, and does not require production of the antibody by any particular method.
  • the term“monoclonal antibody,” as used herein, refers to an antibody that displays a single binding specificity and affinity for a particular epitope or a composition of antibodies in which all antibodies display a single binding specificity and affinity for a particular epitope.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., (1975) Nature 256: 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).
  • the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., (1991) Nature 352: 624-628 and Marks et al., (1991) J. Mol. Biol. 222: 581-597.
  • Antigen binding fragments (including scFvs) of such immunoglobulins are also encompassed by the term“monoclonal antibody” as used herein.
  • Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different epitopes on the antigen, each monoclonal antibody is directed against a single epitope.
  • Monoclonal antibodies can be prepared using any art recognized technique and those described herein such as, for example, a hybridoma method, a transgenic animal, recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), or using phage antibody libraries using the techniques described in, for example, U.S. Patent No. 7,388,088 and PCT Pub. No.
  • Monoclonal antibodies include chimeric antibodies, human antibodies, and humanized antibodies and may occur naturally or be produced recombinantly.
  • a “domain antibody” is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain.
  • two or more V H regions are covalently joined with a peptide linker to create a bivalent domain antibody.
  • the two V H regions of a bivalent domain antibody may target the same or different antigens.
  • bivalent antibody comprises two antigen binding sites. In some instances, the two binding sites have the same antigen specificities. However, bivalent antibodies may be bispecific (see below).
  • single-chain Fv or "scFv” antibody refers to antibody fragments comprising the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker.
  • the monoclonal antibodies herein also include camelized single domain antibodies. See, e.g., Muyldermans etal. (2001) Trends Biochem. Sci. 26:230; Reichmann etal. (1999) J. Immunol. Methods 231:25; WO 94/04678; WO 94/25591; U.S. Pat. No. 6,005,079, which are hereby incorporated by reference in their entireties).
  • the present invention provides single domain antibodies comprising two V H domains with modifications such that single domain antibodies are formed.
  • diabodies refers to small antibody fragments with two antigen binding sites, which fragments comprise a heavy chain variable domain (V H ) connected to a light chain variable domain (V L ) in the same polypeptide chain (V H -V L or V L -V H ).
  • V H heavy chain variable domain
  • V L light chain variable domain
  • the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, e.g., EP 404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448.
  • the antibodies of the present invention also include antibodies with modified (or blocked) Fc regions to provide altered effector functions. See, e.g., U.S. Pat. No. 5,624,821; W 02003/086310; W02005/120571; W02006/0057702; Presta (2006) Adv. Drug Delivery Rev. 58:640-656. Such modification can be used to enhance or suppress various reactions of the immune system, with possible beneficial effects in diagnosis and therapy. Alterations of the Fc region include amino acid changes, such as substitutions, deletions and insertions, glycosylation or deglycosylation, and adding multiple Fc.
  • Changes to the Fc may be utilized to alter the half- life of antibodies in therapeutic antibodies, and a longer half-life would result in less frequent dosing, with the concomitant increased convenience and decreased use of material. See Presta (2005) J. Allergy Clin. Immunol .116:731 at 734-35.
  • Fully human antibody refers to an antibody that comprises human immunoglobulin protein sequences only.
  • a fully human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell.
  • mouse antibody refers to an antibody which comprises mouse immunoglobulin sequences only.
  • hypervariable region refers to the amino acid residues of an antibody that are responsible for antigen-binding.
  • the hypervariable region comprises amino acid residues from a "complementarity determining region” or "CDR” (e.g. residues 24-34 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) in the light chain variable domain and residues 31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) in the heavy chain variable domain; Rabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed.
  • CDR complementarity determining region
  • the term "framework" or "FR" residues refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues.
  • the residue numbering above relates to the Rabat numbering system and does not necessarily correspond in detail to the sequence numbering in the accompanying Sequence Listing.
  • Amino acid residues in antibodies can also be defined using other numbering systems, such as Chothia, enhanced Chothia, IMGT, Kabat/Chothia composite, Honegger (AHo), Contact, or any other conventional antibody numbering scheme.
  • An“isolated antibody,” as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities.
  • isotype refers to the antibody class (e.g., IgG (including IgGl, IgG2, IgG3, and IgG4), IgM, IgA (including IgAl and IgA2), IgD, and IgE antibody) that is encoded by the heavy chain constant region genes of the antibody.
  • IgG including IgGl, IgG2, IgG3, and IgG4
  • IgM including IgAl and IgA2
  • IgD IgD
  • IgE antibody that is encoded by the heavy chain constant region genes of the antibody.
  • effector function refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or a biochemical event that results therefrom.
  • exemplary “effector functions” include Clq binding, complement dependent cytotoxicity (CDC), Fc receptor binding, FcyR- mediated effector functions such as ADCC and antibody dependent cell-mediated phagocytosis (ADCP), and downregulation of a cell surface receptor (e.g., the B cell receptor; BCR).
  • CDC complement dependent cytotoxicity
  • Fc receptor binding FcyR- mediated effector functions
  • ADCP antibody dependent cell-mediated phagocytosis
  • BCR B cell surface receptor
  • Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain).
  • an Fc region refers to the C-terminal region of the heavy chain of an antibody.
  • an Fc region comprises the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CF).
  • epitopes refers to a site on an antigen (e.g., human LAP-TGFp 1 ) to which an immunoglobulin or antibody specifically binds.
  • Epitopes can be formed both from contiguous amino acids (usually a linear epitope) or noncontiguous amino acids juxtaposed by tertiary folding of the protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids in a unique spatial conformation.
  • epitopope mapping refers to the process of identifying the molecular
  • Methods for determining what epitopes are bound by a given antibody include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides from, e.g., LAP-TGFpi are tested for reactivity with a given antibody (e.g., anti-FAP antibody); x-ray crystallography; antigen mutational analysis, two-dimensional nuclear magnetic resonance; yeast display; and hydrogen/deuterium exchange -mass spectrometry (HDX-MS) (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed.
  • HDX-MS hydrogen/deuterium exchange -mass spectrometry
  • the term“binds to the same epitope” with reference to two or more antibodies means that the antibodies bind to the same segment or same segments of amino acid residues, as determined by a given method.
  • Techniques for determining whether antibodies bind to the “same epitope on LAP-TGFp i” with the antibodies described herein include, for example, epitope mapping methods, such as x-ray analyses of crystals of antigen: antibody complexes, which provides atomic resolution of the epitope, and HDX-MS. Other methods monitor the binding of the antibody to antigen fragments (e.g.
  • proteolytic fragments or to mutated variations of the antigen where loss of binding due to a modification of an amino acid residue within the antigen sequence is often considered an indication of an epitope component, such as alanine scanning mutagenesis (Cunningham & Wells (1985) Science 244:1081), yeast display of mutant target sequence variants, or analysis of chimeras (e.g., as described in Examples 2 and 3).
  • an epitope component such as alanine scanning mutagenesis (Cunningham & Wells (1985) Science 244:1081), yeast display of mutant target sequence variants, or analysis of chimeras (e.g., as described in Examples 2 and 3).
  • computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. Antibodies having the same VH and VL or the same CDR1, 2 and 3 sequences are expected to bind to the same epitop
  • Antibodies that“compete with another antibody for binding to a target” refer to antibodies that inhibit (partially or completely) the binding of the other antibody to the target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to a target, may be determined using known binding competition experiments, e.g., BIACORE ® surface plasmon resonance (SPR) analysis. In certain embodiments, an antibody competes with, and inhibits binding of another antibody to a target by at least 50%, 60%, 70%, 80%, 90% or 100%.
  • the level of inhibition or competition may be different depending on which antibody is the“blocking antibody” (i.e., the antibody that when combined with an antigen blocks another immunologic reaction with the antigen).
  • Competition assays can be conducted as described, for example, in Ed Harlow and David Lane, Cold Spring Harb. Protoc. 2006; doi:l0.H0l/pdb.prot4277 or in Chapter 11 of“Using Antibodies” by Ed Harlow and David Lane, Cold Spring Harbor
  • Competing antibodies bind to the same epitope, an overlapping epitope, or to adjacent epitopes ( e.g ., as evidenced by steric hindrance).
  • SPR surface plasmon resonance
  • RIA solid phase direct or indirect radioimmunoassay
  • EIA solid phase direct or indirect enzyme immunoassay
  • sandwich competition assay see Stahli et al., Methods in Enzymology 9:242 (1983)
  • solid phase direct biotin-avidin EIA see Kirkland et al., J. Immunol.
  • solid phase direct labeled assay solid phase direct labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct label RIA using 1-125 label (see Morel et al., Mol. Immunol. 25(l):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA.
  • the terms“specific binding,”“selective binding,”“selectively binds,” and “specifically binds,” refer to antibody binding to an epitope on a predetermined antigen.
  • the antibody (i) binds with an equilibrium dissociation constant (Kp>) of
  • l0 7 M approximately less than 10 8 M, 10 9 M or l0 10 M or even lower when determined by, e.g., surface plasmon resonance (SPR) using a predetermined antigen as the analyte and the antibody as the ligand, or Scatchard analysis of binding of the antibody to antigen positive cells, and (ii) binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen.
  • Any KD greater than about 10 4 M is generally considered to indicate nonspecific binding.
  • KD molar concentration
  • EC50 in the context of an in vitro or in vivo assay using an antibody refers to the concentration of an antibody that induces a response that is 50% of the maximal response, i.e., halfway between the maximal response and the baseline.
  • cross-reacts refers to the ability of an antibody described herein to bind to LAP-TGFP 1 from a different species.
  • an antibody described herein that binds human LAP-TGFpi may also bind another species of LAP-TGFpi (e.g., murine LAP-TGFP 1 , rat FAP-TGFpi, or cynomolgus monkey LAP-TGFP 1 ).
  • Cross -reactivity may be measured by detecting a specific reactivity with purified antigen in binding assays (e.g., SPR, EFISA, bio-layer interferometry) or binding to, or otherwise functionally interacting with, cells physiologically expressing LAP-TGFP 1 (e.g., HT1080 cells overexpressing LAP-TGFP 1 ).
  • binding assays e.g., SPR, EFISA, bio-layer interferometry
  • LAP-TGFP 1 e.g., HT1080 cells overexpressing LAP-TGFP 1
  • Methods for determining cross -reactivity include standard binding assays as described herein, for example, by bio-layer interferometry or flow cytometric techniques.
  • the term“linked” refers to the association of two or more molecules.
  • the linkage can be covalent or non-covalent.
  • the linkage also can be genetic (i.e., recombinantly fused). Such linkages can be achieved using a wide variety of art recognized techniques, such as chemical conjugation and recombinant protein production.
  • nucleic acid molecule is intended to include DNA molecules and RNA molecules.
  • a nucleic acid molecule may be single- stranded or double- stranded, but preferably is double- stranded DNA.
  • isolated nucleic acid molecule as used herein in reference to nucleic acids encoding antibodies or antibody fragments (e.g., VH, VL, CDR3), is intended to refer to a nucleic acid molecule in which the nucleotide sequences are essentially free of other genomic nucleotide sequences, e.g., those encoding antibodies that bind antigens other than FAP, which other sequences may naturally flank the nucleic acid in human genomic DNA.
  • vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g ., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as“recombinant expression vectors” (or simply,“expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonly used form of vector.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • conservative sequence modifications include conservative nucleotide and amino acid substitutions, as well as, nucleotide and amino acid additions and deletions.
  • modifications can be introduced into a sequence in a table herein (e.g., Table 34) by standard techniques known in the art, such as site-directed mutagenesis and PCR- mediated mutagenesis.
  • Conservative amino acid substitutions include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g.
  • a predicted nonessential amino acid residue in an anti-LAP antibody is preferably replaced with another amino acid residue from the same side chain family.
  • Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well-known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).
  • mutations can be introduced randomly along all or part of an anti-LAP antibody coding sequence, such as by saturation mutagenesis, and the resulting modified anti-LAP antibodies can be screened for binding activity.
  • nucleic acids the term“substantial homology” indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, usually at least about 80% to 85%, 85% to 90% or 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides.
  • substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.
  • the term“substantial homology” indicates that two polypeptides, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate amino acid insertions or deletions, in at least about 80% of the amino acids, usually at least about 80% to 85%, 85% to 90%, 90% to 95%, and more preferably at least about 98% to 99.5% of the amino acids.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
  • the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a
  • the percent identity between two nucleotide or two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ( J. Mol. Biol.
  • nucleic acid and protein sequences described herein can further be used as a“query sequence” to perform a search against public databases to, for example, identify related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(l7):3389-3402.
  • the default parameters of the respective programs e.g ., XBLAST and NBLAST
  • recombinant host cell (or simply“host cell”), as used herein, is intended to refer to a cell that comprises a nucleic acid that is not naturally present in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term“host cell” as used herein.
  • inhibitor refers to any statistically significant decrease in biological activity, including partial and full blocking of the activity.
  • “inhibition” can refer to a statistically significant decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% in biological activity.
  • TGFpi activation refers to the release of the mature cytokine TGFpi from the latent complex made up of LAP and TGFP 1.
  • TGFP 1 activation There are many mechanisms known to induce TGFP 1 activation (see Robertson IB, Rifkin DB. Unchaining the beast; insights from structural and evolutionary studies on TGFP 1 secretion, sequestration, and activation. Cytokine Growth Factor Rev. 2013 Aug;24(4):355-72).
  • the mature cytokine can be detected using a specific ELISA or similar detection methodology or through the use of a reporter cell line that expresses a TGFP receptor.
  • the term“inhibits TGFP 1 activation” includes any measurable decrease in TGFP 1 activation, e.g., an inhibition of TGFpl activation by at least about 10%, for example, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99%, or about 100%, relative to a control (e.g., a control antibody).
  • the inhibition may be specific to a single mechanism of TGFpi activation or may be generalizable to all mechanisms of TGFpl activation.
  • the term“inhibits TGFP 1 activation” includes inhibition of at least one activation mechanism.
  • the terms“treat,”“treating,” and“treatment,” as used herein, refer to therapeutic or preventative measures described herein.
  • the methods of“treatment” employ administration to a subject with a tumor or cancer or a subject who is predisposed to having such a disease or disorder, an anti-LAP antibody (e.g., anti-human LAP antibody) described herein, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • an anti-LAP antibody e.g., anti-human LAP antibody
  • Immunotherapy refers to the treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response.
  • Immuno stimulating therapy or“immunostimulatory therapy” refers to a therapy that results in increasing (inducing or enhancing) an immune response in a subject for, e.g., treating cancer.
  • immune cell refers to the subset of blood cells known as white blood cells, which include mononuclear cells such as lymphocytes, monocytes, macrophages, and granulocytes.
  • immunosuppressive cell refers to a cell that contributes to or promotes an immunosuppressive tumor microenvironment. The presence of a population of
  • immunosuppressive cells in a tumor microenvironment increases the tumor's resistance to an immune response, resulting in tumor protection, tumor escape, and/or tumor metastasis. Unless countered in some manner, these immunosuppressive cells can decrease the efficacy of immune- mediated anti-cancer treatments.
  • immunosuppressive cells include cancer-associated fibroblasts, myeloid-derived suppressor cells, regulatory T cells (Tregs), tumor cells expressing LAP, and immunosuppressive macrophages.
  • Tregs e.g., CD4, FoxP3, CD127, and CD25
  • macrophages e.g., CSF-IR, CD203, CD206, CD163, IL-10, and TGFp
  • cancer associated fibroblasts e.g., alpha smooth muscle actin, fibroblast activation protein, tenascin-C, periostin, NG2, vimentin, desmin, PDGFR alpha and beta, FSP-l, ASPN, and STC1
  • myeloid-derived suppressor cells e.g., CDl lb, CD33, CD14, or CD15, and low levels of HLA DR.
  • immunosuppressive cells may also be important in suppressing the immune system in other disease states.
  • suppressive T cells refer to T cells that contribute to or promote an immunosuppressive microenvironment.
  • exemplary suppressive T cells include CD4+ regulatory T cells and CD8+ regulatory T cells.
  • Such cells can be identified by one skilled in the art using, e.g., flow cytometry to identify markers such as FoxP3, LAP or Helios.
  • Tregs refer to immunosuppressive cells that generally suppress or downregulate induction and proliferation of effector T cells.
  • Tregs generally express the biomarkers CD4, FOXP3, and CD25 and are thought to be derived from the same lineage as naive CD4 cells.
  • T effector (“T eff ”) cells refers to T cells (e.g., CD4+ and CD8+ T cells) with cytolytic activities as well as T helper (Th) cells, which secrete inflammatory cytokines and activate and direct other immune cells, but does not include regulatory T cells (Treg cells).
  • administering refers to the physical introduction of a molecule (e.g., an antibody or antigen binding fragment that binds LAP) or of a composition comprising a therapeutic agent (e.g., an anti-LAP antibody or antigen binding fragment) to a subject, using any of the various methods and delivery systems known to those skilled in the art.
  • a molecule e.g., an antibody or antigen binding fragment that binds LAP
  • a composition comprising a therapeutic agent
  • Preferred routes of administration for antibodies described herein include intravenous, intraperitoneal,
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion, as well as in vivo electroporation.
  • an antibody described herein can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally,
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • cancer refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may result in the formation of malignant tumors or cells that invade neighboring tissues and may metastasize to distant parts of the body through the lymphatic system or bloodstream.
  • autoimmune disease describes a disease state or syndrome whereby a subject's body produces a dysfunctional immune response against the subject's own body components, with adverse effects.
  • fibrosis refers to disorders or disease states that are caused by or accompanied by the abnormal deposition of extracellular matrix (i.e., not formation of fibrous tissue in normal organ and tissue). Fibrosis is characterized by excessive accumulation of extracellular matrix in the affected tissue that often results in destruction of its normal architecture and causes significant organ dysfunction. Although fibrotic conditions in various organs have diverse etiologies, fibrosis typically results from chronic persistent inflammation induced by a variety of stimuli, such as chronic infections, ischemia, allergic and autoimmune reactions, chemical insults or radiation injury (from Biemacka, 2011 Growth Factors. 2011 Oct;29(5): 196-202. doi: 10.3109/08977194.2011.595714. Epub 2011 Jul 11). Fibrosis may affect the heart, liver, kidney, lung and skin and is also a central feature in many cancers.
  • cell therapy refers to a method of treatment involving the administration of live cells (e.g., CAR T cells, and NK cells).
  • live cells e.g., CAR T cells, and NK cells.
  • treat refers to any type of intervention or process performed on, or administering an active agent (e.g., an anti-LAP antibody or antigen binding fragment) to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, or slowing down or preventing the progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease.
  • Treatment can be of a subject having a disease or a subject who does not have a disease (e.g., for prophylaxis).
  • “adjunctive” or“combined” administration includes simultaneous administration of the agents and/or compounds in the same or different dosage form, or separate administration of the compounds (e.g., sequential administration).
  • at least one agent comprises an anti-LAP antibody or antigen binding fragment.
  • a first antibody or antigen binding fragment e.g., an anti-LAP antibody or antigen binding fragment
  • a second, third, or more antibodies or antigen binding fragments can be simultaneously administered in a single formulation.
  • the first and second (or more) antibodies or antigen binding fragments can be formulated for separate administration and are administered concurrently or sequentially.
  • Combination therapy means administration of two or more therapeutic agents in a coordinated fashion, and includes, but is not limited to, concurrent dosing.
  • combination therapy encompasses both co-administration (e.g. administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and serial or sequential administration, provided that administration of one therapeutic agent is conditioned in some way on administration of another therapeutic agent.
  • one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a prescribed period of time.
  • the anti-LAP antibody can be administered first followed by (e.g. , immediately followed by) the administration of a second antibody (e.g., an anti-PD-l antibody) or antigen binding fragment, or vice versa.
  • the anti-LAP antibody or antigen binding fragment is administered prior to administration of the second antibody or antigen binding fragment. In another embodiment, the anti-LAP antibody or antigen binding fragment is administered, for example, a few minutes (e.g., within about 30 minutes) or at least one hour of the second antibody or antigen binding fragment. Such concurrent or sequential administration preferably results in both antibodies or antigen binding fragments being simultaneously present in treated patients.
  • an effective dose or“effective dosage” is defined as an amount sufficient to achieve or at least partially achieve a desired effect.
  • a "therapeutically effective amount” or “therapeutically effective dosage” of a drug is any amount of the drug or therapeutic agent that, when used alone or in combination with another therapeutic agent, promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase or therapeutic agent in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
  • a therapeutically effective amount or dosage of a drug or therapeutic agent includes a
  • prophylactically effective amount or a “prophylactically effective dosage” which is any amount of the drug or therapeutic agent that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or of suffering a recurrence of disease, inhibits the development or recurrence of the disease.
  • the ability of a therapeutic agent to promote disease regression or inhibit the development or recurrence of the disease can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.
  • the administration of effective amounts of the anti-LAP antibody or antigen binding fragment alone, or anti-LAP antibody or antigen binding fragment combined with another compound or agent can result in at least one therapeutic effect, including, for example, reduced tumor growth or size, reduced number of indicia of cancer (e.g., metastatic lesions) appearing over time, complete remission, partial remission, or stable disease.
  • an immune checkpoint blocker such as an anti-PD-l antibody
  • CBR complete remission
  • PR partial remission
  • SD stable disease
  • a therapeutically effective amount or dosage of the drug or therapeutic agent inhibits tumor cell growth by at least about 20%, by at least about 30% by at least about 40%, by at least about 50%, by at least about 60%, by at least above 70%, by at least about 80%, or by at least about 90% relative to untreated subjects.
  • a therapeutically effective amount or dosage of the drug or therapeutic agent completely inhibits cell growth or tumor growth, i.e., inhibits cell growth or tumor growth by 100%.
  • the ability of a compound or therapeutic agent, including an antibody, to inhibit tumor growth can be evaluated using the assays described herein. Alternatively, this property of a composition comprising the compound or therapeutic agent can be evaluated by examining the ability of the composition to inhibit cell growth; such inhibition can be measured in vitro by assays known to the skilled practitioner.
  • patient includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.
  • the term“subject” includes any human or non-human animal.
  • the methods and compositions described herein can be used to treat a subject having cancer.
  • the term“non-human animal” includes all vertebrates, e.g., mammals and non mammals, such as non-human primates, sheep, cats, dogs, cows, chickens, amphibians, reptiles, etc.
  • sample refers to tissue, bodily fluid, or a cell (or a fraction of any of the foregoing) taken from a patient or a subject. Normally, the tissue or cell will be removed from the patient, but in vivo diagnosis is also contemplated.
  • a tissue sample can be taken from a surgically removed tumor and prepared for testing by conventional techniques.
  • lymphomas and leukemias lymphocytes, leukemic cells, or lymph tissues can be obtained (e.g., leukemic cells from blood) and appropriately prepared.
  • Other samples including urine, tears, serum, plasma, cerebrospinal fluid, feces, sputum, cell extracts etc. can also be useful for particular cancers.
  • “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other.
  • the term“and/or” as used in a phrase such as“A and/or B” includes“A and B,”“A or B,”“A” alone, and“B” alone.
  • the term“and/or” as used in a phrase such as“A, B, and/or C” encompasses each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.
  • ug and uM are used interchangeably with “pg” and “pM,” respectively.
  • an isolated anti-LAP antibody i.e., an antibody that binds LAP
  • an antibody that binds LAP or antigen binding fragment thereof.
  • an isolated anti-LAP antibody e.g., recombinant humanized, chimeric, or human antibody
  • antigen binding fragment thereof which comprises:
  • a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 162, 172, and 164, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 165, 166, and 167, respectively;
  • a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 162, 173, and 164, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 165, 166, and 167, respectively;
  • the anti-LAP antibody e.g., recombinant humanized, chimeric, or human antibody
  • antigen binding fragment comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 19, 20, and 21, respectively, except wherein position 56 of the heavy chain variable region (corresponding to position 7 of SEQ ID NO: 17) is an amino acid other than N (e.g., Q, S, H, L, D)) or is substituted with an amino acid residue other than N (e.g., Q, S, H, L, D).
  • N e.g., Q, S, H, L, D
  • the anti-LAP antibody e.g., recombinant humanized, chimeric, or human antibody
  • antigen binding fragment comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 54, 55, and 56, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 57, 58, and 59, respectively, except wherein position 54 of the heavy chain variable region (corresponding to position 5 of SEQ ID NO: 55) is an amino acid other than N (e.g., Q, A, H, S) or is substituted with an amino acid residue other than N (e.g., Q, A, H, S).
  • the anti-LAP antibody e.g., recombinant humanized, chimeric, or human antibody
  • antigen binding fragment comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 54, 55, and 56, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 57, 58, and 59, respectively, except wherein position 102 of the heavy chain variable region (corresponding to position 4 of SEQ ID NO: 56) is an amino acid other than D (e.g., A, E, G) ) or is substituted with an amino acid residue other than D (e.g., A, E, G).
  • the anti-LAP antibody e.g., recombinant humanized, chimeric, or human antibody
  • antigen binding fragment comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 54, 55, and 56, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 57, 58, and 59, respectively, except wherein position 54 of the heavy chain variable region (corresponding to position 5 of SEQ ID NO: 55) is an amino acid other than N (e.g., Q, A, H, S) or is substituted with an amino acid residue other than N (e.g., Q, A, H, S), and wherein position 102 of the heavy chain variable region (corresponding to position 4 of SEQ ID NO: 56) is an amino acid other than D (e.g., A, E, G) ) or is substituted with an amino acid residue other than
  • the anti-LAP antibody e.g., recombinant humanized, chimeric, or human antibody
  • antigen binding fragment comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 110, 111, and 112, respectively, and a light chain variable region comprising CDR1,
  • CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 113, 114, and 115, respectively, except wherein position 54 of the heavy chain variable region (corresponding to position 5 of SEQ ID NO: 111) is an amino acid other than N (e.g., Q, G, A, S, H, L, D)) or is substituted with an amino acid residue other than N (e.g., Q, G, A, S, H, L, D).
  • N e.g., Q, G, A, S, H, L, D
  • the anti-LAP antibody e.g., recombinant humanized, chimeric, or human antibody
  • antigen binding fragment comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 162, 163, and 164, respectively, and a light chain variable region comprising CDR1,
  • CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 165, 166, and 167, respectively, except wherein position 56 of the heavy chain variable region (corresponding to position 7 of SEQ ID NO: 163) is an amino acid other than N (e.g., Q, G, A, S, H, L, D) or is substituted with an amino acid residue other than N (Q, G, A, S, H, L, D).
  • N e.g., Q, G, A, S, H, L, D
  • the anti-LAP antibody e.g., recombinant humanized, chimeric, or human antibody
  • antigen binding fragment comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 179, 180, and 181, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 182, 183, and 184, respectively, except wherein position 55 of the heavy chain variable region (corresponding to position 6 of SEQ ID NO: 180) is an amino acid other than N (e.g., Q, G, A, S, H, L, D) or is substituted with an amino acid residue other than N (e.g., Q, G, A, S, H, L, D).
  • N e.g., Q, G, A, S, H, L, D
  • the anti-LAP antibody (e.g., recombinant humanized, chimeric, or human antibody) comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 225, 226, and 227, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 228, 229, and 230, respectively, except wherein position 55 of the heavy chain variable region (corresponding to position 5 of SEQ ID NO: 226) is an amino acid other than D (e.g., G, A, E) or is substituted with an amino acid residue other than D (e.g., G, A, E).
  • D e.g., G, A, E
  • the anti-LAP antibody comprises the heavy chain CDR sequences of any of subparts (a)-(ap) above, and a constant region, e.g., a human IgG constant region (e.g., IgGl, IgG2, IgG3, or IgG4, or variants thereof).
  • the constant region is a human IgGl constant region comprising the amino acid sequence set forth in SEQ ID NO: 196.
  • the constant region is a variant human IgG4 constant region comprising the amino acid sequence set forth in SEQ ID NO: 197.
  • a heavy chain variable region comprising the heavy chain CDR sequences of any of subparts (a)-(ap) above may be linked to a constant domain to form a heavy chain (e.g., a full length heavy chain).
  • a light chain variable region comprising the light chain CDR sequences of any of subparts (a)-(ap) above may be linked to a constant region to form a light chain (e.g., a full length light chain).
  • a full length heavy chain (with the exception of the C-terminal lysine (K) or with the exception of the C-terminal glycine and lysine (GK), which may be absent or removed) and full length light chain combine to form a full length antibody.
  • isolated anti-LAP antibodies comprising: (a) heavy and light chain variable region sequences comprising SEQ ID NOs: 42 and 52, respectively;
  • the anti-LAP antibody has variable region sequences with potential liability sites, e.g., deamidation sites and/or isomerization sites) removed.
  • the anti-LAP antibody comprises heavy and light chain variable region sequences of any of subparts (a)-(d) above, except wherein position 56 of the heavy chain variable region is an amino acid other than N (e.g., Q, S, H, L, D)) or is substituted with an amino acid residue other than N (e.g., Q, S, H, L, D).
  • position 56 of the heavy chain variable region is an amino acid other than N (e.g., Q, S, H, L, D)) or is substituted with an amino acid residue other than N (e.g., Q, S, H, L, D).
  • the anti-LAP antibody comprises heavy and light chain variable region sequences of any of subparts (e)-(l) above, except wherein position 54 of the heavy chain variable region is an amino acid other than N (e.g., Q, A, H, S) or is substituted with an amino acid residue other than N (e.g., Q, A, H, S).
  • position 54 of the heavy chain variable region is an amino acid other than N (e.g., Q, A, H, S) or is substituted with an amino acid residue other than N (e.g., Q, A, H, S).
  • the anti-LAP antibody comprises heavy and light chain variable region sequences of any of subparts (e)-(l) above, except wherein position 102 of the heavy chain variable region is an amino acid other than D (e.g., A, E, G) or is substituted with an amino acid residue other than D (e.g., A, E, G).
  • the anti-LAP antibody comprises heavy and light chain variable region sequences of any of subparts (e)-(l) above, except wherein position 54 of the heavy chain variable region is an amino acid other than N (e.g., Q, A, H, S) or is substituted with an amino acid residue other than N (e.g., Q, A, H, S), and wherein position 102 of the heavy chain variable region is an amino acid other than D (e.g., A, E, G) or is substituted with an amino acid residue other than D (e.g., A, E, G).
  • position 54 of the heavy chain variable region is an amino acid other than N (e.g., Q, A, H, S) or is substituted with an amino acid residue other than N (e.g., Q, A, H, S)
  • position 102 of the heavy chain variable region is an amino acid other than D (e.g., A, E, G) or is substituted with an amino acid residue other than D (e.g., A, E, G).
  • the anti-LAP antibody comprises heavy and light chain variable region sequences of any of subparts (m)-(t) above, except wherein position 54 of the heavy chain variable region is an amino acid other than N (e.g., Q, G, A, S, H, L, D)) or is substituted with an amino acid residue other than N (e.g., Q, G, A, S, H, L, D).
  • position 54 of the heavy chain variable region is an amino acid other than N (e.g., Q, G, A, S, H, L, D)) or is substituted with an amino acid residue other than N (e.g., Q, G, A, S, H, L, D).
  • the anti-LAP antibody comprises a heavy chain and/or light chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the heavy chain and/or light chain variable region sequences of any of subparts (a)-(t) above.
  • the heavy chain and/or light chain variable region sequences of any of subparts (a)-(t) above has 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 amino acid substitutions (e.g., conservative amino acid substitutions).
  • the anti-LAP antibody does not have heavy and light chain variable region sequences which are identical to SEQ ID NOs: 22 and 23, respectively; 60 and 61, respectively; 116 and 117, respectively; 168 and 169, respectively; or 185 and 186, respectively.
  • anti-LAP antibodies can be tested for various properties that are clinically advantageous (e.g., binding to LAP-TGPpl, inhibiting the activation of T ⁇ RbI, binding to various cell (e.g., immune cell) populations, inhibiting tumor growth in vivo) using the assays and animal models described herein, for example, in the Examples.
  • the anti-LAP antibody comprises the heavy chain variable region sequences of any of subparts (a)-(t) above, and a constant region, e.g., a human IgG constant region (e.g., IgGl, IgG2, IgG3, or IgG4, or variants thereof).
  • a human IgG constant region e.g., IgGl, IgG2, IgG3, or IgG4, or variants thereof.
  • the constant region is a human IgGl constant region comprising the amino acid sequence set forth in SEQ ID NO: 196.
  • the constant region is a variant human IgG4 constant region comprising the amino acid sequence set forth in SEQ ID NO: 197.
  • the heavy chain variable region sequences of any of subparts (a)-(t) above may be linked to a constant domain to form a heavy chain (e.g., a full length heavy chain).
  • the light chain variable region sequences of any of subparts (a)-(t) above may be linked to a constant region to form a light chain (e.g., a full length light chain).
  • a full length heavy chain (with the exception of the C-terminal lysine (K) or with the exception of the C-terminal glycine and lysine (GK), which may be absent or removed) and full length light chain combine to form a full length antibody.
  • anti-LAP antibodies comprising
  • antibodies comprising heavy and light chain variable region sequences comprising (a) SEQ ID NOs: 234 and 224, respectively, (b) SEQ ID NOs: 235 and 224, respectively, or (c) SEQ ID NOs: 236 and 224, respectively.
  • anti-LAP antibodies comprising heavy and light chain sequences comprising (a) SEQ ID NOs: 237 and 222, respectively, (b) SEQ ID NOs: 238 and 222, respectively, or (c) SEQ ID NOs: 239 and 222, respectively.
  • isolated anti-LAP antibodies comprising:
  • the full length heavy chain lacks the C-terminal lysine residue (which may be absent or removed).
  • the anti-LAP antibody has heavy and light chain sequences with potential liability sites, e.g., deamidation sites and/or isomerization sites) removed.
  • the anti-LAP antibody comprises heavy and light chain sequences of any of subparts (a)-(g) above, except wherein position 56 of the heavy chain is an amino acid other than N (e.g., Q, S, H, L, D) or is substituted with an amino acid residue other than N (e.g., Q, S, H, L, D).
  • position 56 of the heavy chain is an amino acid other than N (e.g., Q, S, H, L, D) or is substituted with an amino acid residue other than N (e.g., Q, S, H, L, D).
  • the anti-LAP antibody comprises heavy and light chain sequences of any of subparts (h)-(w) above, except wherein position 54 of the heavy chain is an amino acid other than N (e.g., Q, A, H, S) or is substituted with an amino acid residue other than N, H, or S (e.g., Q, A, H, S).
  • position 54 of the heavy chain is an amino acid other than N (e.g., Q, A, H, S) or is substituted with an amino acid residue other than N, H, or S (e.g., Q, A, H, S).
  • the anti-LAP antibody comprises heavy and light chain sequences of any of subparts (h)-(w) above, except wherein position 102 of the heavy chain variable region is an amino acid other than D (e.g., A, E, G) or is substituted with an amino acid residue other than D (e.g., A, E, G).
  • the anti-LAP antibody comprises heavy and light chain sequences of any of subparts (h)-(w) above, except wherein position 54 of the heavy chain is an amino acid other than N (e.g., Q, A, H, S) or is substituted with an amino acid residue other than N, H, or S (e.g., Q, A, H, S) and position 102 of the heavy chain variable region is an amino acid other than D (e.g., A, E, G) or is substituted with an amino acid residue other than D (e.g., A, E, G).
  • position 54 of the heavy chain is an amino acid other than N (e.g., Q, A, H, S) or is substituted with an amino acid residue other than N, H, or S (e.g., Q, A, H, S)
  • position 102 of the heavy chain variable region is an amino acid other than D (e.g., A, E, G) or is substituted with an amino acid residue other than D (e.g., A, E,
  • the anti-LAP antibody comprises heavy and light chain sequences of any of subparts (x)-(ai) above, except wherein position 54 of the heavy chain variable region is an amino acid other than N (e.g., Q, G, A, S, H, L, D) or is substituted with an amino acid residue other than N (e.g., Q, G, A, S, H, L, D).
  • the anti-LAP antibody comprises a heavy chain and/or light chain sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.7% identical to the heavy chain and/or light chain sequences of any of subparts (a)-(ai) above.
  • the heavy chain and/or light chain sequences of any of subparts (a)-(ai) above has 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 amino acid substitutions (e.g., conservative amino acid substitutions).
  • the anti-LAP antibody does not have a heavy and/or light chain variable region sequence which is identical to SEQ ID NOs: 24 and 25, respectively; 62 and 63 respectively; or 118 and 119, respectively.
  • anti-LAP antibodies can be tested for various properties that are clinically advantageous (e.g., binding to LAP-TGFP 1 , inhibiting the activation of TGFpl, binding to various cell (e.g., immune cell) populations, inhibiting tumor growth in vivo) using the assays and animal models described herein, for example, in the Examples.
  • an anti-LAP antibody or antigen binding fragment comprising VHCDR1-3 sequences of SEQ ID NOs: 110, 120, and 113, respectively, and VLCDR1-3 sequences of SEQ ID NOs: 113, 114, and 115, respectively, has one or more amino acid substitutions in the CDRs or variable regions.
  • amino acid substitutions for example, in some embodiments, no more than 3 amino acids (i.e., 1, 2, or 3 amino acids) are substituted in the six heavy and light chain CDRs (collectively), or two heavy and light chain variable regions (collectively).
  • an anti-LAP antibody or antigen binding fragment comprises a VHCDR1 which has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, for example, conservative amino acid substitutions, relative to GYTFTSYWMH (SEQ ID NO: 110).
  • an anti-LAP antibody or antigen binding fragment comprises a VHCDR2 which has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, for example, conservative amino acid substitutions, relative to RIDPQSGGIK (SEQ ID NO: 120).
  • the VHCDR2 comprises the sequence: RX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X9, wherein X 1 -X9 can be any amino acid. In some embodiments, only 1 position among X 1 -X9 is substituted relative to the amino acid sequence of SEQ ID NO: 120.
  • an anti-LAP antibody or antigen binding fragment comprises a VHCDR3 comprising 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, for example, conservative amino acid substitutions, relative to WDYGGYFDV (SEQ ID NO: 112).
  • the VHCDR3 comprises the sequence: WX1YGGYFX2X3 (SEQ ID NO: 242), wherein X1-X3 can be any amino acid. In some embodiments, only 1 position among X1-X3 is substituted relative to the amino acid sequence of SEQ ID NO: 112.
  • an anti-LAP antibody or antigen binding fragment comprises a VLCDR1 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions, for example, conservative amino acid substitutions, relative to RASQDITNYLN (SEQ ID NO: 113).
  • the VLCDR1 may comprise the sequence: RX1X2X3DIX4X5YX6X7, wherein Xi- X 7 is any amino acid. In some embodiments, only 1 position among X1-X7 is substituted relative to the amino acid sequence of SEQ ID NO: 113.
  • an anti-LAP antibody or antigen binding fragment comprises a VLCDR2 comprising 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions, for example, conservative amino acid substitutions, relative to YTSRLHS (SEQ ID NO: 114).
  • the VLCDR2 comprises the sequence: YX1X2RX3X4X5, wherein X1-X5 is any amino acid. In some embodiments, only 1 position among X1-X5 is substituted relative to the amino acid sequence of SEQ ID NO: 114.
  • an anti-LAP antibody or antigen binding fragment comprises a VLCDR3 comprising 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, for example, conservative amino acid substitutions, relative to QQGDTLPWT (SEQ ID NO: 115).
  • the VLCDR3 may comprise the sequence: QQGDXiLPWT (SEQ ID NO: 243), wherein Xi is any amino acid.
  • the anti-LAP antibody or antigen binding fragment described herein binds to LAP-TGFp 1 (e.g., human LAP-TGFp 1 ) in the absence of an anchor protein.
  • LAP-TGFp 1 e.g., human LAP-TGFp 1
  • the anti-LAP antibody or antigen binding fragment described herein binds to recombinant human LAP-TGFp 1 in an assay that does not include an anchor protein.
  • the anti-LAP antibody or antigen binding fragment described herein binds to LAP-TGFp 1 (e.g., soluble LAP-TGFp 1 ) with a K D of 100 nM or less, such as 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, such as 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, 1 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, 0.1 nM or less, 10 nM to 0.1 nM, 5 nM to 0.1 nM, 3 nM to 0.1 nM, 1
  • the anti- LAP antibody or antigen binding fragment described herein binds to LAP-TGFpi (e.g., human, cyno, rat and ) with a KD in an Example herein.
  • LAP-TGFpi e.g., human, cyno, rat and
  • the anti-LAP antibody or antigen binding fragment described herein binds to human LAP-TGFpl, rat LAP-TGFP 1 , cyno LAP-TGFp 1 , and/or murine LAP-TGFpi .
  • the anti-LAP antibody or antigen binding fragment described herein described herein binds to LAP-TGFpi complexed with an anchor protein on
  • the anchor protein is GARP or LRRC33.
  • the anti-LAP antibody or antigen binding fragment described herein described herein selectively inhibits TGFP 1 activation on immunosuppressive cells without inhibiting TGFpi activation on extracellular matrix.
  • the anti-LAP antibody or antigen binding fragment described herein does not bind to LAP complexed with LTBP1, LTBP3, and/or LTBP4.
  • the anti-LAP antibody or antigen binding fragment described herein does not bind to LAP-TGFP2 (e.g., human LAP-TGFP2) and LAP-TGFP3 (e.g., human LAP-TGFp3), as assessed by, e.g., flow cytometry using cells that overexpress TGFP2 or TGFP3, or bio-layer interferometry with recombinant LAP-TGFP2 or LAP-TGFp3.
  • LAP-TGFP2 e.g., human LAP-TGFP2
  • LAP-TGFP3 e.g., human LAP-TGFp3
  • the anti-LAP antibody or antigen binding fragment described herein binds to LAP-TGFP2 or LAP-TGFP3 with a signal or affinity that is not significantly above the signal seen with a control antibody (e.g., isotype control) or the signal seen in the absence of anti-LAP antibody (e.g., as described in Example 2).
  • a control antibody e.g., isotype control
  • the signal seen in the absence of anti-LAP antibody e.g., as described in Example 2.
  • the anti-LAP antibody or antigen binding fragment described herein inhibits TGFP 1 activation, as assessed by, e.g., ELISA detection of free TGFP 1 in a culture of P3U1 cells overexpressing LAP-TGFP 1.
  • the anti-LAP antibody or antigen binding fragment described herein inhibits (or is determined to inhibit) TGFpl activation by about 50% or more, e.g., by about 60% or more, by about 70% or more, by about 80% or more, or by about 90% or more, as assessed by ELISA, e.g., ELISA detection of free T ⁇ Eb 1 in a culture of P3U1 cells overexpressing LAP-TGEP 1 (e.g., as described in
  • the anti-LAP antibody or antigen binding fragment described herein binds to mouse and human LAP-TGEP 1 , as assessed by, e.g., flow cytometry of activated immune cell populations.
  • the anti-LAP antibody or antigen binding fragment described herein does not bind to free TGFpl (i.e., TGFpl without LAP), as assessed by, e.g., ELISA. In some embodiments, the anti-LAP antibody or antigen binding fragment described herein does not bind to empty LAP (i.e., LAP that is not complexed with TGFpl), as assessed by, e.g., bio layer interferometry.
  • the anti-LAP antibody or antigen binding fragment described herein binds to free T ⁇ Eb 1 or empty with a signal or affinity that is not significantly above the signal seen with a control antibody (e.g., isotype control) or the signal seen in the absence of anti-LAP antibody (e.g., as described in Example 2).
  • a control antibody e.g., isotype control
  • the signal seen in the absence of anti-LAP antibody e.g., as described in Example 2.
  • the anti-LAP antibody or antigen binding fragment described herein binds to human LAP-TGEP 1 comprising K27C and Y75C mutations (SEQ ID NO: 12. In another embodiment, the anti-LAP antibody or antigen binding fragment described herein does not bind to (or are determined not to bind to) human LAP-TGEpi comprising a Y74T mutation (SEQ ID NO: 13). In another embodiment, the anti-LAP antibody or antigen binding fragment described herein binds to (or is determined to bind to) human LAP-TGEpi comprising K27C and Y75C mutations, but not to LAP-TGEpi comprising a Y74T mutation.
  • the anti-LAP antibodies bind to all or a portion of residues 82-130 of human LAP-TGFpl (SEQ ID NO: 1).
  • the anti-LAP antibodies bind within residues 82-130 of human LAP-TGFp 1 (SEQ ID NO: l).
  • the anti-LAP antibody or antigen binding fragment binds to one or more regions on human LAP-TGEP 1 (SEQ ID NO: 1) comprising or consisting of amino acids 31-40, 274-280, and 340-343.
  • the anti-LAP antibody or antigen binding fragment binds to amino acids 31-40, 274-280, and 340-343 of human LAP-TGEpi (SEQ ID NO: 1).
  • the epitope is determined by cryo- EM.
  • the anti-LAP antibody or antigen binding fragment binds to one or more regions on human an LAP-TGEpi (SEQ ID NO: 1) comprising or consisting of amino acids 31-38, 278-281, and 342-344. In some embodiments, the anti-LAP antibodies bind to amino acids 31-38, 278-281, and 342-344 of human LAP-TGFpl (SEQ ID NO: 1).
  • the epitope is determined by cryo-EM.
  • the anti-LAP antibody or antigen binding fragment binds to one or more regions on human an LAP-TGEP 1 (SEQ ID NO: 1) comprising or consisting of amino acids 35-43, 272-275, 280-283, and 340 (SEQ ID NO: 1).
  • the anti-LAP antibody or antigen binding fragment binds to amino acids 35-43, 272-275, 280-283, and 340 of human LAP-TGFpl (SEQ ID NO: 1).
  • the epitope is determined by cryo-EM.
  • the anti-LAP antibody or antigen binding fragment described herein binds to LAP-TGEp i on cells, such as immune cells, e.g., immunosuppressive cells.
  • Immunosuppressive cells include, but are not limited to, suppressive T cells (e.g., regulatory T cells, activated T cells, suppressive CD8+ T cells), Ml macrophages, M2 macrophages, dendritic cells, regulatory B cells, granulocytic MDSCs, and/or monocytic MDSCs, as assessed, e.g., by flow cytometry.
  • suppressive T cells e.g., regulatory T cells, activated T cells, suppressive CD8+ T cells
  • Ml macrophages e.g., M2 macrophages, dendritic cells, regulatory B cells, granulocytic MDSCs, and/or monocytic MDSCs
  • the anti-LAP antibody or antigen binding fragment described herein binds to cells other than immune cells, such as tumor cells, fibroblasts
  • the anti-LAP antibody or antigen binding fragment described herein binds to LAP-TGFP 1 on both immune cells (e.g., immunosuppressive cells) and non-immune cells.
  • the anti-LAP antibody or antigen binding fragment described herein binds to LAP-TGEP 1 on GARP-positive cells (e.g., GARP-positive immunosuppressive cells). In some embodiments, the anti-LAP antibody or antigen binding fragment described herein binds to (or are determined to bind to) LAP-TGEP 1 on GARP-negative cells (e.g., GARP- negative immunosuppressive cells). In some embodiments, the anti-LAP antibody or antigen binding fragment described herein binds to LAP-TGEP 1 on both GARP-positive and GARP- negative cells, as assessed, e.g., by flow cytometry.
  • the anti-LAP antibody or antigen binding fragment described herein reduces the endogenous expression of CD73. In some embodiments, the anti-LAP antibody or antigen binding fragment described herein inhibits the increase of CD73 expression caused by a treatment, e.g., radiation.
  • CD73 expression can be determined using standard methods known in the art (e.g., as described in Example 16).
  • the anti-LAP antibody or antigen binding fragment described herein binds to LAP-TGFP 1 expressed on cells (e.g., human or mouse LAP-TGFp i expressed on, e.g., P3U1 cells) with an EC50 of 1000 ng/ml or less, 500 ng/ml or less, 200 ng/ml or less,
  • 150 ng/ml or less 100 ng/ml or less, 50 ng/ml or less, 25 ng/ml or less, 10 ng/ml or less, 5 ng/ml or less, 2 ng/ml or less, 1 ng/ml to 200 ng/ml, 1 ng/ml to 150 ng/ml, 1 ng/ml to 100 ng/ml, 1 ng/ml to 50 ng/ml, 1 ng/ml to 25 ng/ml, 1 ng/ml to 10 ng/ml, or 1 ng/ml to 5 ng/ml, as measured by flow cytometry (e.g., as described in Example 2).
  • the binding of the anti-LAP antibody or antigen binding fragment to LAP-TGFP 1 may also be defined using quantitative immunofluorescence by flow cytometry, which allows the number of antibody molecules bound per cell to be quantified. Accordingly, in some
  • the number of anti-LAP antibodies bound to a cell that also expresses GARP may be equal to the number of anti-GARP antibodies bound to that cell, or may be at least 80%, at least 50%, at least 20%, at least 10%, at least 5%, at least 1%, or at least 0.1% of the number of anti-GARP antibodies bound to that cell.
  • the number of LAP-TGFp i molecules expressed per cell may be quantified using quantitative immunofluorescence using an anti-LAP antibody of a group that detects the majority of LAP molecules; examples of such antibodies include 2F8, 2C9, 16B4 and the anti-LAP monoclonal antibody #27232 (R&D Systems).
  • the number of anti-LAP antibodies bound to the cell may be equal to the number of LAP molecules on the cell, or may be at least 80%, at least 50%, at least 20%, at least 10%, at least 5%, at least 1% or at least 0.1% of the number of LAP molecules expressed on that cell.
  • the anti-LAP antibody or antigen binding fragment described herein inhibits TGFP 1 activation by, for example, 10% or more, for example, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more, relative to a control (e.g., a control antibody), as measured by ELISA (e.g., as described in Example 4).
  • a control e.g., a control antibody
  • ELISA e.g., as described in Example 4
  • the anti-LAP antibody or antigen binding fragment described herein binds to soluble LAP-TGFp 1 with high affinity, for example, with a KD of 10 7 M or less, 10 8 M or less, 10 9 M or less, 10 10 M or less, 10 11 M or less, 10 12 M or less, 10 12 M to 10 7 M, 10 11 M to 10 7 M, 10 10 M to 10 7 M, or 10 9 M to 10 7 M, as measured by bio-layer interferometry ( e.g ., as described in Example 1).
  • a KD 10 7 M or less, 10 8 M or less, 10 9 M or less, 10 10 M or less, 10 11 M or less, 10 12 M or less, 10 12 M to 10 7 M, 10 11 M to 10 7 M, 10 10 M to 10 7 M, or 10 9 M to 10 7 M, as measured by bio-layer interferometry ( e.g ., as described in Example 1).
  • the anti-LAP antibody or antigen binding fragment described herein does not bind to LAP-TGFp 1 in the extracellular matrix.
  • the anti-LAP antibody or antigen binding fragment described herein do not bind to LAP-TGFP 1 in the extracellular matrix, as assessed by ELISA, wherein the O.D. signal for the antibody or antigen binding fragment binding is not significantly above the signal seen in the absence of the anti- LAP antibody or antigen binding fragment described herein or the signal seen with a control antibody (e.g., isotype control) (e.g., as described in Example 5).
  • a control antibody e.g., isotype control
  • the anti-LAP antibody or antigen binding fragment described herein do not inhibit TGFP activation in the ECM, as assessed by, e.g., ELISA detection of free TGFp 1 in an assay combining a source of LAP-TGFp 1 in the ECM (e.g., as described in Example 5) with MMP-2, MMP-9, thrombospondin or cells expressing anb6 or anb8 integrins.
  • the anti-LAP antibody or antigen binding fragment described herein binds to EAR-TORb 1 on platelets.
  • at least 5%, at least 10%, at least 20% or at least 50% of platelets can be detected by binding of the anti-LAP antibody (e.g. display a signal above that seen with an isotype control antibody) by flow cytometry (e.g., as described in Example 6).
  • the anti-LAP antibody or antigen binding fragment described herein binds to platelets but do not cause platelet aggregation or platelet degranulation.
  • the anti-LAP antibody or antigen binding fragment described herein binds to immune cells, e.g., suppressive T cells (e.g., regulatory T cells), M2
  • the anti-LAP antibody e.g. display a signal above that seen with an isotype control antibody
  • flow cytometry e.g., as described in Example 7
  • the anti-LAP antibody or antigen binding fragment described herein is considered to bind to these cell types if they bind >2 standard deviations above isotype control.
  • the anti-LAP antibody or antigen binding fragment described herein binds to GARP-negative leukocytes.
  • at least 0.5% , at least 1%, at least 2%, at least 5%, at least 7%, at least 10%, at least 20% or at least 50% of GARP-negative leukocytes can be detected by binding of the anti-LAP antibody (e.g. display a signal above that seen with an isotype control antibody) by flow cytometry (e.g., as described in Example 7).
  • an antibody or antigen binding fragment that exhibits one or more of the functional properties described above will be understood to relate to a statistically significant difference in the particular activity relative to that seen in the absence of the antibody (e.g., or when a control antibody of irrelevant specificity is present).
  • the anti-LAP antibody-induced increases in a measured parameter effects a statistically significant increase by at least 10% of the measured parameter, more preferably by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% (i.e., 2 fold), 3 fold, 5 fold or 10 fold.
  • anti-LAP antibody-induced decreases in a measured parameter effects a statistically significant decrease by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, ,99%, or 100%.
  • anti-LAP antibodies that bind to the same epitope on human LAP-TGPpl as any of the anti-LAP antibodies described herein. These antibodies have the ability to cross-compete for binding to human LAP-TGPpl with any of the anti-LAP antibodies described herein. In some embodiments, the anti-LAP antibodies bind one or more amino acids within residues 82-130 of human LAP-TGPpl (SEQ ID NO: 1).
  • Antibodies disclosed herein include all known forms of antibodies and other protein scaffolds with antibody-like properties.
  • the antibody can be a human antibody, a humanized antibody, a bispecific antibody, an immunoconjugate, a chimeric antibody, or a protein scaffold with antibody-like properties, such as fibronectin or ankyrin repeats.
  • the antibody is a bispecific antibody comprising a first and second binding region, wherein the first binding region comprises the binding specificity (e.g., antigen binding region) of an anti-LAP antibody described herein, and a second binding region that does not bind to LAP.
  • the second binding region binds to a protein that is not expressed on platelets.
  • the antibody also can be a Lab, P(ab’) 2 , scLv, ALPIBODY, avimer, nanobody, single chain antibody, or a domain antibody.
  • the antibody also can have any isotype, including any of the following isotypes: IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgAsec, IgD, and IgE.
  • Full- length antibodies can be prepared from VH and VL sequences using standard recombinant DNA techniques and nucleic acid encoding the desired constant region sequences to be operatively linked to the variable region sequences.
  • the antibodies described herein may have effector function or may have reduced or no effector function.
  • anti-LAP antibodies comprise an effector-less or mostly effector-less Fc, e.g., IgG2 or IgG4.
  • variable regions described herein may be linked to an Fc comprising one or more modification, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
  • an antibody described herein may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, to alter one or more functional properties of the antibody.
  • the numbering of residues in the Fc region is that of the EU index of Kabat.
  • the Fc region is a variant Fc region, e.g., an Fc sequence that has been modified (e.g., by amino acid substitution, deletion and/or insertion) relative to a parent Fc sequence (e.g., an unmodified Fc polypeptide that is subsequently modified to generate a variant), to provide desirable structural features and/or biological activity.
  • modifications can be made in the Fc region in order to generate an Fc variant that (a) has increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased or decreased complement mediated cytotoxicity (CDC), (c) has increased or decreased affinity for Clq and/or (d) has increased or decreased affinity for a Fc receptor relative to the parent Fc.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement mediated cytotoxicity
  • c has increased or decreased affinity for Clq
  • d has increased or decreased affinity for a Fc receptor relative to the parent Fc.
  • Such Fc region variants will generally comprise at least one amino acid modification in the Fc region. Combining amino acid modifications is thought to be particularly desirable.
  • the variant Fc region may include two, three, four, five, etc. substitutions therein, e.g. of the specific Fc region positions identified herein.
  • a variant Fc region may also comprise a sequence alteration wherein amino acids involved in disulfide bond formation are removed or replaced with other amino acids. Such removal may avoid reaction with other cysteine-containing proteins present in the host cell used to produce the antibodies described herein. Even when cysteine residues are removed, single chain Fc domains can still form a dimeric Fc domain that is held together non-covalently.
  • the Fc region may be modified to make it more compatible with a selected host cell. For example, one may remove the PA sequence near the N-terminus of a typical native Fc region, which may be recognized by a digestive enzyme in E. coll such as proline
  • one or more glycosylation sites within the Fc domain may be removed. Residues that are typically glycosylated (e.g ., asparagine) may confer cytolytic response. Such residues may be deleted or substituted with unglycosylated residues (e.g., alanine).
  • sites involved in interaction with complement such as the Clq binding site, may be removed from the Fc region. For example, one may delete or substitute the EKK sequence of human IgGl.
  • sites that affect binding to Fc receptors may be removed, preferably sites other than salvage receptor binding sites. In other
  • an Fc region may be modified to remove an ADCC site.
  • ADCC sites are known in the art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) with regard to ADCC sites in IgGl. Specific examples of variant Fc domains are disclosed for example, in PCT Publication numbers WO 97/34631 and WO 96/32478.
  • the hinge region of Fc is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased.
  • This approach is described further in U.S. Patent No. 5,677,425 by Bodmer et al. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding.
  • SpA Staphylococcyl protein A
  • the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody.
  • one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody.
  • the effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.
  • one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • the Fc region may be modified to increase antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity for an Fey receptor by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278,
  • ADCC antibody dependent cellular cytotoxicity
  • Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E.
  • Exemplary variants include 239D/332E, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F, 267E/324T, and 267E/268F/324T.
  • Fc modifications that increase binding to an Fey receptor include amino acid
  • Fc modifications that can be made to Fes are those for reducing or ablating binding to FcyR and/or complement proteins, thereby reducing or ablating Fc-mediated effector functions such as ADCC, ADCP, and CDC.
  • Exemplary modifications include but are not limited substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, and 328, wherein numbering is according to the EU index.
  • Exemplary substitutions include but are not limited to 234G, 235G, 236R, 237K, 267R, 269R, 325L, and 328R, wherein numbering is according to the EU index.
  • An Fc variant may comprise 236R/328R.
  • the Fc region may comprise a non-naturally occurring amino acid residue at additional and/or alternative positions known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; PCT Patent Publication numbers WO 00/42072; WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752; WO 04/074455; WO 04/099249; WO 04/063351; WO 05/070963; WO 05/040217, WO 05/092925 and WO 06/020114).
  • Fc variants that enhance affinity for an inhibitory receptor FcyRllb may also be used. Such variants may provide an Fc fusion protein with immunomodulatory activities related to FcyRllb + cells, including for example B cells and monocytes. In one embodiment, the Fc variants provide selectively enhanced affinity to FcyRllb relative to one or more activating receptors. Modifications for altering binding to FcyRllb include one or more modifications at a position selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, and 332, according to the EU index.
  • Exemplary substitutions for enhancing FcyRllb affinity include but are not limited to 234D, 234E, 234F, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E.
  • Exemplary substitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y.
  • Fc variants for enhancing binding to FcyRllb include 235Y/267E, 236D/267E, 239D/268D, 239D/267E, 267E/268D, 267E/268E, and 267E/328F.
  • the antibody is modified to increase its biological half-life.
  • this may be done by increasing the binding affinity of the Fc region for FcRn.
  • one or more of more of following residues can be mutated: 252, 254, 256, 433, 435, 436, as described in U.S. Pat. No. 6,277,375.
  • Specific exemplary substitutions include one or more of the following: T252F, T254S, and/or T256F.
  • the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos.
  • exemplary variants that increase binding to FcRn and/or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, including for example 2591, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M.
  • Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428L, 428F, 250Q/428L (Hinton et al., 2004, J. Biol. Chem.
  • hybrid IgG isotypes with particular biological characteristics may be used.
  • an IgGl/IgG3 hybrid variant may be constructed by substituting IgGl positions in the CH2 and/or CH3 region with the amino acids from IgG3 at positions where the two isotypes differ.
  • a hybrid variant IgG antibody may be constructed that comprises one or more substitutions, e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F.
  • an IgGl/IgG2 hybrid variant may be constructed by substituting IgG2 positions in the CH2 and/or CH3 region with amino acids from IgGl at positions where the two isotypes differ.
  • a hybrid variant IgG antibody may be constructed that comprises one or more substitutions, e.g., one or more of the following amino acid substitutions: 233E, 234L, 235L, -236G (referring to an insertion of a glycine at position 236), and 327A.
  • Fc mutants include: S298A/E333A/L334A, S239D/I332E, S239D/I332E/A330L, L235V/F243L/R292P/Y300L/ P396L, and M428L/N434S.
  • the glycosylation of an antibody is modified.
  • an aglycoslated antibody can be made (i.e ., the antibody lacks glycosylation).
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen.
  • carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for antigen.
  • Glycosylation of the constant region on N297 may be prevented by mutating the N297 residue to another residue, e.g., N297A, and/or by mutating an adjacent amino acid, e.g., 298 to thereby reduce glycosylation on N297.
  • an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures.
  • altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies described herein to thereby produce an antibody with altered glycosylation.
  • PCT Publication number WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al. (2002) J. Biol. Chem. 277:26733-26740).
  • PCT Publication number WO 99/54342 by Umana et al.
  • glycoprotein-modifying glycosyl transferases e.g ., beta(l,4)-N-acetylglucosaminyltransferase III (GnTIII)
  • GnTIII glycoprotein-modifying glycosyl transferases
  • an antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody.
  • the antibody, or fragment thereof typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment.
  • PEG polyethylene glycol
  • the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
  • polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.
  • the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies described herein. See for example, European patent number EP 0 154 316 by Nishimura et al. and European patent number EP 0 401 384 by Ishikawa et al.
  • the affinities and binding properties of an Fc region for its ligand may be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art including, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration).
  • in vitro assay methods biochemical or immunological based assays
  • equilibrium methods e.g., enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA)
  • kinetics e.g., BIACORE analysis
  • indirect binding assays e.g., competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis
  • These and other methods may utilize a label on one or more of the components being examined and/or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels.
  • detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels.
  • Anti-LAP antibodies which bind to the same or similar epitopes to the antibodies disclosed herein (and thus also cross-compete with the antibodies disclosed herein) may be raised using immunization protocols.
  • the resulting antibodies can be screened for high affinity binding to human LAP-TGEpi .
  • Selected antibodies can then be studied, e.g., in yeast display assay in which sequence variants of LAP-TGEpi are presented on the surface of yeast cells, or by hydrogen-deuterium exchange experiments, to determine the precise epitope bound by the antibody.
  • Antibodies which bind to the same epitope as the anti-LAP antibodies described herein can also be generated using chimeric constructs, e.g., chicken-human chimeras of LAP-TGFpl. Since human and chicken sequences can be combined to yield a LAP-TGEpi protein that folds correctly (as described in Example 2), the method can be used to generate immunogens to specific epitopes of interest on LAP-TGEpi . With this strategy, the majority of the sequence would be taken from chicken LAP-TGEP 1 , with small sections of human LAP-TGEpi inserted in regions containing the desired epitope.
  • Exemplary epitopes on LAP-TGEP 1 that can be targeted using this strategy include, for example, the lower arm of LAP-TGEP 1 , the latency loop of LAP-TGFpl, or an epitope comprising amino acids 82-130 of human LAP-TGEP 1.
  • Example 3 Exemplary chicken-human chimera constructs are described in Example 3. This chimeric protein could be used to immunize chickens to yield monoclonal antibodies. Since the chicken LAP-TGFp 1 would be recognized as self, the immune response will be focused on the human sequence. Antibodies generated using this approach can be tested for various combinations of factors.
  • functions/properties e.g., binding to LAP-TGFpl, inhibiting T ⁇ Eb 1 activation, binding to ECM, binding to cells such as immunosuppressive cells
  • standard methods known in the art, e.g., the methods described herein.
  • the epitope to which an antibody binds can be determined using art-recognized methods.
  • An anti-LAP antibody is considered to bind to the same epitope as a reference anti-LAP antibody if it, e.g., contacts one or more of the same residues on human LAP-TGFpi as the reference antibody; contacts one or more of the same residues within at least one region of human LAP- TGFp 1 as the reference antibody; contacts a majority of residues within at least one region of human LAP-TGFpi as the reference antibody; contacts a majority of the same residues within each region of human LAP-TGFP 1 as the reference antibody; contacts a majority of the same residues along the entire length of human LAP-TGFpi as the reference antibody; contacts all of the same distinct regions of human LAP-TGFpi as the reference antibody; contacts all of the same residues at any one region on human LAP-TGFP 1 as the reference antibody; or contacts all of the same residues at all of the same regions of human LAP
  • Techniques for determining antibodies that bind to the“same epitope on human LAP- TGFp i” with the anti-LAP antibodies described herein include x-ray analyses of crystals of antigen: antibody complexes, which provides atomic resolution of the epitope. Other methods monitor the binding of the antibody to antigen fragments or mutated variations of the antigen where loss of binding due to an amino acid modification within the antigen sequence indicates the epitope component. Methods may also rely on the ability of an antibody of interest to affinity isolate specific short peptides (either in native three-dimensional form or in denatured form) from combinatorial phage display peptide libraries or from a protease digest of the target protein. The peptides are then regarded as leads for the definition of the epitope corresponding to the antibody used to screen the peptide library. For epitope mapping, computational algorithms have also been developed that have been shown to map conformational discontinuous epitopes.
  • the epitope or region comprising the epitope can also be identified by screening for binding to a series of overlapping peptides spanning human LAP-TGFP 1.
  • the method of Jespers et al. (1994) Biotechnology 12:899 may be used to guide the selection of antibodies having the same epitope and therefore similar properties to the anti-LAP antibodies described herein.
  • the heavy chain of the anti-LAP antibody is paired with a repertoire of (e.g., human) light chains to select a LAP-binding antibody, and then the new light chain is paired with a repertoire of (e.g., human) heavy chains to select a (e.g., human) LAP-binding antibody having the same epitope or epitope region as an anti-LAP antibody described herein.
  • variants of an antibody described herein can be obtained by mutagenesis of cDNA sequences encoding the heavy and light chains of the antibody.
  • the epitope or epitope region (an“epitope region” is a region comprising the epitope or overlapping with the epitope) bound by a specific antibody may also be determined by assessing binding of the antibody to peptides comprising LAP-TGEpi fragments.
  • a series of overlapping peptides encompassing the LAP-TGEP 1 sequence may be synthesized and screened for binding, e.g. in a direct ELISA, a competitive ELISA (where the peptide is assessed for its ability to prevent binding of an antibody to LAP-TGEpi bound to a well of a microtiter plate), or on a chip.
  • Such peptide screening methods may not be capable of detecting some discontinuous functional epitopes.
  • An epitope may also be identified by MS-based protein footprinting, such as HDX-MS and Fast Photochemical Oxidation of Proteins (FPOP), structural methods such as X-ray crystal structure determination, molecular modeling, and nuclear magnetic resonance spectroscopy.
  • MS-based protein footprinting such as HDX-MS and Fast Photochemical Oxidation of Proteins (FPOP)
  • FPOP Fast Photochemical Oxidation of Proteins
  • SP-Cryo-EM Single particle cryo electron microscopy
  • SP-Cryo-EM is a technique for macromolecular structure analysis which uses a high intensity electron beam to image biological specimens in their native environment at cryogenic temperature.
  • SP-cryo-EM has emerged as a complementary technique to crystallography and NMR for determining near-atomic level structures suitable for application in drug discovery (Renaud et al. Nat Rev Drug Discov 2018;17:471-92; Scapin et al. Cell Chem Biol 2018;25:1318-25; Ceska et al. Biochemical Society Transactions 2019: p. BST20180267).
  • SP- Cryo-EM has the further advantage of allowing access to larger and more complex biological systems, with the possibility of characterizing multiple conformational or compositional solution states from the same sample, providing insights into more biologically relevant states of the macromolecule.
  • a small volume of sample e.g., 3 pl aliquot
  • the frozen grid is then loaded into the microscope and hundreds to thousands of images of different areas of the grids are collected.
  • These images contain two-dimensional projections of the biological macromolecule (particles): using mathematical tools and GPU powered algorithms, the particles are identified, extracted, and classified; in the subsequent step, the different classes are used to compute one or more 3D reconstructions, corresponding to different conformations, oligomerization or binding states if they coexist in the same sample. The individual reconstructions can then be refined to high resolution.
  • nucleic acid molecules that encode the anti-LAP antibodies or antigen binding fragments described herein.
  • the nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • a nucleic acid described herein can be, for example, DNA or RNA and may or may not contain intronic sequences.
  • the nucleic acid is a cDNA molecule.
  • the nucleic acids described herein can be obtained using standard molecular biology techniques.
  • hybridomas e.g ., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below
  • cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques.
  • nucleic acid encoding the antibody can be recovered from the library.
  • nucleic acid molecules that encode the VH and/or VL sequences, or heavy and/or light chain sequences, of any of the anti-LAP antibodies or antigen binding fragments described herein.
  • Host cells comprising the nucleotide sequences (e.g., nucleic acid molecules) described herein are encompassed herein.
  • DNA fragments encoding VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene.
  • VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.
  • operatively linked is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.
  • the isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (hinge, CH1, CH2 and/or CH3).
  • heavy chain constant regions hinge, CH1, CH2 and/or CH3.
  • the sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL.
  • the sequences of human light chain constant region genes are known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the light chain constant region can be a kappa or lambda constant region.
  • nucleic acid molecules with conservative substitutions that do not alter the resulting amino acid sequence upon translation of the nucleic acid molecule.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies described herein can be made using the hybridoma method first described by Kohler et al. , Nature, 256:495 (1975), or any later developments thereof, or by recombinant DNA methods (U.S. Pat. No. 4,816,567).
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al , Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988); Hammer-ling, et al.
  • Human antibodies can be made by a variety of methods known in the art, including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publication numbers WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, the contents of which are herein incorporated by reference in their entireties. Human antibodies can also be produced using transgenic mice which express human immunoglobulin genes, and upon immunization are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For an overview of this technology for producing human antibodies, see, Lonberg and Huszar, 1995, Int. Rev. Immunol. 13:65-93.
  • Phage display technology (McCafferty et al., Nature 348:552-553 (1990)) also can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.
  • Human antibodies can also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275, the contents of which are herein incorporated by reference in their entireties).
  • Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection.”
  • a selected non-human monoclonal antibody e.g., a mouse antibody
  • Chimeric antibodies can be prepared based on the sequence of a murine monoclonal antibody.
  • DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and engineered to contain non-murine (e.g.,. human)
  • the murine variable regions can be linked to human constant regions using methods known in the art (see e.g., U.S. Patent No. 4,816,567 to Cabilly et al.).
  • Humanized forms of anti-LAP antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies are typically human immunoglobulins (recipient antibody) in which residues from a CDR or hypervariable region of the recipient are replaced by residues from a CDR or hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies can comprise residues which are not found in the recipient antibody or in the donor antibody.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor antibody CDR or the consensus framework can be mutagenized by substitution, insertion and/or deletion of at least one amino acid residue so that the CDR or framework residue at that site does not correspond exactly to either the donor antibody or the consensus framework.
  • the term "consensus framework” refers to the framework region in the consensus immunoglobulin sequence.
  • the term "consensus immunoglobulin sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see e.g., Winnaker, From Genes to Clones (Veriagsgesellschaft, Weinheim, Germany 1987). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. Where two amino acids occur equally frequently, either can be included in the consensus sequence.
  • “Vernier zone” refers to a subset of framework residues that may adjust CDR structure and fine-tune the fit to antigen as described by Foote and Winter (1992, J. Mol. Biol.
  • Vernier zone residues form a layer underlying the CDRs and can impact on the structure of CDRs and the affinity of the antibody.
  • Human immunoglobulin (Ig) sequences that can be used as a recipient are well known in the art.
  • Framework residues in the human framework regions can be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Three- dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Antibodies can be humanized using a variety of techniques known in the art, including, but not limited to, those described in Jones et ah, Nature 321 :522 (1986); Verhoeyen et ah, Science 239: 1534 (1988), Sims et al., J. Immunol. 151 : 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al, Proc. Natl. Acad. Sci. U.S. A. 89:4285 (1992); Presta et ah, J. Immunol. 151 :2623 (1993), Padlan,
  • the anti-LAP antibodies generated using the methods described above can be tested for desired functions, such as particular binding specificities, binding affinities, targeted cell populations, using methods known in the art and described in the Examples, for example, art- recognized protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays.
  • An aspect of the invention provides molecules that may be used to screen for an antibody or antigen binding fragment that binds LAP, a complex comprising LAP, and/or a complex comprising LAP-TGEpi .
  • the molecules in Table 4 i.e., molecules having the amino acid sequence of any of SEQ ID NO: 1, and 198-210) are used to screen or determine binding of at least one binding protein.
  • Exemplary assays include, but are not limited to, immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA), FACS, enzyme-linked immunoabsorbent assay (ELISA), bio-layer interferometry (e.g., ForteBio assay), and Scatchard analysis.
  • an in vitro binding assay such as radioimmunoassay (RIA), FACS, enzyme-linked immunoabsorbent assay (ELISA), bio-layer interferometry (e.g., ForteBio assay), and Scatchard analysis.
  • framework residues within the variable domains of the parental monoclonal antibody e.g., to improve the properties of the antibody or fragment.
  • framework modifications are made to decrease the immunogenicity of the antibody or fragment.
  • analogous residues from the immune repertoire of the species in which the antibody is to be used e.g., human residues in the case of human therapeutics.
  • Such an antibody or fragment is referred to as a "humanized” antibody or fragment.
  • One approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody or fragment that has undergone somatic mutation can contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody or fragment framework sequences to the germline sequences from which the antibody or fragment is derived.
  • Another approach is to revert to the original parental (e.g., rodent) residue at one or more positions of the engineered (e.g. humanized) antibody, e.g. to restore binding affinity that may have been lost in the process of replacing the framework residues.
  • the engineered (e.g. humanized) antibody e.g. to restore binding affinity that may have been lost in the process of replacing the framework residues.
  • the anti-LAP antibodies and antigen -binding fragments thereof are engineered (e.g., humanized) to include modifications in the framework and/or CDRs to improve their properties.
  • engineered changes can be based on molecular modeling.
  • a molecular model for the variable region for the parental (non-human) antibody sequence can be constructed to understand the structural features of the antibody and used to identify potential regions on the antibody that can interact with the antigen.
  • Conventional CDRs are based on alignment of immunoglobulin sequences and identifying variable regions. Rabat et al., (1991) Sequences of Proteins of Immunological Interest, Rabat, et al.; National Institutes of Health, Bethesda, Md.
  • the molecular model for the variable region of the non-human antibody can be used to guide the selection of regions that can potentially bind to the antigen.
  • the potential antigen binding regions based on model differ from the conventional“CDR”s or“hyper variable” loops.
  • Commercial scientific software such as MOE (Chemical Computing
  • Human frameworks can be selected based on best matches with the non-human sequence both in the frameworks and in the CDRs.
  • FR4 framework 4 in VH
  • VJ regions for the human germlines are compared with the corresponding non-human region.
  • FR4 (framework 4) in VF J-kappa and J- Fambda regions of human germline sequences are compared with the corresponding non human region.
  • the CDRs are grafted into the selected human frameworks.
  • certain residues in the VF-VH interface can be retained as in the non-human (parental) sequence.
  • Molecular models can also be used for identifying residues that can potentially alter the CDR conformations and hence binding to antigen. In some cases, these residues are retained as in the non-human
  • Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as “deimmunization” and is described in further detail in U.S. Patent No. 7,125,689.
  • the deamidation of asparagine may occur on NG, DG, NG, NS, NA,
  • the antibodies of the present disclosure do not contain deamidation or
  • an asparagine (Asn) residue may be
  • the asparagine is changed to glutamine (Gln). It may also be desirable to alter an amino acid adjacent to an asparagine (Asn) or glutamine (Gln) residue to reduce the likelihood of deamidation, which occurs at greater rates when small amino acids occur adjacent to asparagine or glutamine. See, Bischoff & Kolbe (1994) J. Chromatog. 662:261.
  • any methionine residues (typically solvent exposed Met) in CDRs may be changed to Lys, Leu, Ala, or Phe or other amino acids in order to reduce the possibility that the methionine sulfur would oxidize, which could reduce antigen -binding affinity and also contribute to molecular heterogeneity in the final antibody preparation. Id.
  • antibodies e.g ., humanized antibodies
  • antigen-binding fragments thereof disclosed herein e.g ., antibody 20E6 and humanized versions thereof and antibody and 28 Gl 1 and humanized versions thereof
  • antibody 20E6 and humanized versions thereof and antibody and 28 Gl 1 and humanized versions thereof can also be engineered to
  • modifications within the Fc region typically to alter one or more properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or effector function (e.g., antigen-dependent cellular cytotoxicity).
  • the antibody typically to alter one or more properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or effector function (e.g., antigen-dependent cellular cytotoxicity).
  • antibodies and antigen-binding fragments thereof disclosed herein can be chemically modified (e.g., one or more
  • chemical moieties can be attached to the antibody) or be modified to alter its
  • glycosylation again to alter one or more properties of the antibody or fragment.
  • the numbering of residues in the Fc region is that of the EU index of Kabat.
  • antibodies and antigen-binding fragments thereof disclosed herein also include antibodies and fragments with
  • modified (or blocked) Fc regions to provide altered effector functions. See, e.g., U.S.
  • Alterations of the Fc region include amino acid changes (substitutions, deletions and insertions), glycosylation or deglycosylation, and adding multiple Fc regions.
  • the antibody or antigen-binding fragment of the invention is an IgG4 isotype antibody or fragment comprising a Serine to Proline mutation at a position corresponding to position 228 (S228P; EU index) in the hinge region of the heavy chain constant region.
  • S228P Serine to Proline mutation at a position corresponding to position 228
  • This mutation has been reported to abolish the heterogeneity of inter-heavy chain disulfide bridges in the hinge region (Angal et al. supra ⁇ , position 241 is based on the Kabat numbering system).
  • the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is increased or decreased. This approach is described further in U.S. Patent No. 5,677,425.
  • the number of cysteine residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
  • the Fc hinge region of an antibody or antigen-binding fragment of the invention is mutated to decrease the biological half-life of the antibody or fragment. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc- hinge fragment such that the antibody or fragment has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding.
  • SpA Staphylococcyl protein A
  • invention e.g., antibody 20E6 and humanized versions thereof and antibody 20E6 and humanized versions thereof
  • antibody 20E6 and humanized versions thereof is modified to increase its biological half-life.
  • T252L, T254S, T256F as described in U.S. Patent No. 6,277,375.
  • the antibody can be altered within the
  • the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody or antigen-binding fragment.
  • one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand and retains the antigen-binding ability of the parent antibody.
  • the effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260.
  • amino acids selected from amino acid residues are selected from amino acid residues
  • 329, 331 and 322 can be replaced with a different amino acid residue such that the
  • amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement.
  • the Fc region is modified to decrease the ability of the antibody or antigen-binding fragment of the invention (e.g ., antibody 20E6 and humanized versions thereof and antibody 20E6 and humanized versions thereof) to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to decrease the affinity of the antibody or fragment for an Fey receptor by modifying one or more amino acids at the following positions: 238, 239, 243, 248, 249, 252, 254, 255, 256, 258, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298,
  • the Fc region is modified to decrease the ability of the antibody of the invention (e.g., antibody 20E6 and humanized versions thereof) to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243 and 264.
  • the Fc region of the antibody or fragment is modified by changing the residues at positions 243 and 264 to alanine.
  • the Fc region is modified to decrease the ability of the antibody or fragment to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243, 264, 267 and 328.
  • the Fc region of an anti-LAP antibody is modified to increase or reduce the ability of the antibody or antigen-binding fragment to mediate effector function and/or to increase/decrease their binding to the Fcgamma receptors (FcyRs).
  • FcyRs Fcgamma receptors
  • FcR FcgammaRI
  • FcgammaRII CD64
  • FcgammaRII CD32
  • FcgammaRIII CD 16
  • the Fc receptor is also important for antibody cross-linking, which can be important for anti-tumor immunity.
  • an antigen binding protein of the present invention can be assessed for ADCC
  • such mutations are in one or more of positions selected from 239, 332 and 330 (IgGl), or the equivalent positions in other IgG isotypes.
  • suitable mutations are S239D and I332E and A330L.
  • the antigen binding protein of the invention herein described is mutated at positions 239 and 332, for example S239D and I332E or in a further embodiment it is mutated at three or more positions selected from 239 and 332 and 330, for example S239D and I332E and A330L. (EU index numbering).
  • an antibody comprising a heavy chain constant region with an altered glycosylation profile such that the antigen binding protein has enhanced effector function.
  • the antibody has enhanced ADCC or enhanced CDC or wherein it has both enhanced
  • ADCC and CDC effector function ADCC and CDC effector function.
  • suitable methodologies to produce antigen binding proteins with an altered glycosylation profile are described in PCT Publication numbers W02003011878 and W02006014679 and European patent number EP1229125.
  • the present invention provides“non-fucosylated” or “afucosylated” antibodies.
  • Non-fucosylated antibodies harbor a tri-mannosyl core structure of complex-type N-glycans of Fc without fucose residue.
  • glycoengineered antibodies that lack core fucose residue from the Fc N-glycans may exhibit stronger ADCC than fucosylated equivalents due to enhancement of
  • the present invention also provides a method for the production of an antibody according to the invention comprising the steps of: a) culturing a
  • the recombinant host cell comprising an expression vector comprising the isolated nucleic acid as described herein, wherein the recombinant host cell does not comprise an alpha- l,6-fucosyltransferase; and b) recovering the antigen binding protein.
  • the recombinant host cell may not normally contain a gene encoding an alpha- 1,6- fucosyltransferase (for example yeast host cells such as Pichia sp.) or may have been genetically modified to inactivate an alpha- l,6-fucosyltransferase.
  • Recombinant host cells which have been genetically modified to inactivate the FUT8 gene encoding an alpha- l,6-fucosyltransferase are available. See, e.g., the POTELLIGENTTM
  • the antibodies or antigen-binding fragments of the invention comprise a particular glycosylation pattern.
  • an afucosylated or an aglycosylated antibody or fragment can be made (i.e ., the antibody lacks fucose or glycosylation, respectively).
  • the glycosylation pattern of an antibody or fragment may be altered to, for example, increase the affinity or avidity of the antibody or fragment for a
  • LAP antigen Such modifications can be accomplished by, for example, altering one or more of the glycosylation sites within the antibody or fragment sequence.
  • amino acid substitutions can be made that result in
  • variable region framework glycosylation sites removal of one or more of the variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity or avidity of the antibody or fragment for antigen. See, e.g., U.S. Patent
  • Antibodies and antigen-binding fragments disclosed herein may further include those produced in lower eukaryote host cells, in particular fungal host cells such as yeast and filamentous fungi have been genetically engineered to produce glycoproteins that have mammalian- or human-like glycosylation patterns (See for example, Choi et al, (2003) Proc. Natl. Acad. Sci. 100: 5022-5027; Hamilton el al.,
  • glycoproteins that are produced in the cells such that compositions of glycoproteins can be produced wherein a particular N-glycan structure predominates (see, e.g., U.S. Patent No. 7,029,872 and U.S. Patent No. 7,449,308).
  • These genetically modified host cells have been used to produce antibodies that have predominantly particular /V-glycan structures (See for example, Li et al., (2006) Nat. Biotechnol. 24: 210-215).
  • the antibodies and antigen-binding fragments thereof disclosed herein further include those produced in lower eukaryotic host cells and which comprise fucosylated and non- fucosylated hybrid and complex /V-glycans, including bisected and multiantennary species, including but not limited to /V-glycans such as GlcNAc (i-4) Man 3 GlcNAc 2 ; Galp- 4 ) GlcNAc (!- 4) Man 3 GlcNAc 2 ; NANA (i-4) Gal (i-4) GlcNAc (i-4) Man 3 GlcNAc 2 .
  • the antibodies and antigen-binding fragments are antibodies and antigen-binding fragments.
  • antibody 20E6 and humanized versions thereof may comprise antibodies or fragments having at least one hybrid /V-glycan selected from the group consisting of GlcNAcMansGlcNAc 2 ; GalGlcNAcMansGlcNAc 2 ; and
  • the hybrid /V-glycan is the predominant /V-glycan species in the composition.
  • the antibodies and antigen-binding fragments thereof provided herein comprise antibodies and fragments having at least one complex /V-glycan selected from the group consisting of GlcNAcMan 3 GlcNAc 2 ;
  • GalGlcNAcMan GlcNAc 2 NANAGalGlcNAcMan GlcNAc 2 ; GlcNAc 2 Man GlcNAc 2 ; GalGlcN Ac 2 Man GlcN Ac 2 ; Gal 2 GlcNAc 2 Man GlcNAc 2 ;
  • the complex /V-glycan are the predominant /V-glycan species in the composition.
  • the complex /V-glycan is a particular /V-glycan species that comprises about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex N- glycans in the composition.
  • the antibody and antigen binding fragments thereof provided herein comprise complex N-glycans, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex /V-glycans comprise the structure NANA 2 Gal 2 GlcNAc 2 Man 3 GlcNAc 2 , wherein such structure is afucosylated.
  • complex N-glycans comprise the structure NANA 2 Gal 2 GlcNAc 2 Man 3 GlcNAc 2 , wherein such structure is afucosylated.
  • Such structures can be produced, e.g., in engineered Pichia pastoris host cells.
  • the /V-glycan is fucosylated.
  • the fucose is in an al, 3-linkage with the GlcNAc at the reducing end of the /V-glycan, an al, 6-linkage with the GlcNAc at the reducing end of the /V-glycan, an al, 2-linkage with the Gal at the non reducing end of the /V-glycan, an al, 3-linkage with the GlcNac at the non-reducing end of the /V-glycan, or an al, 4-linkage with a GlcNAc at the non-reducing end of the /V-glycan.
  • the glycoform is in an al, 3-linkage or al, 6-linkage fucose to produce a glycoform selected from the group consisting of MansGlcNAc 2 (Fuc), GlcNAcMansGlcNAc 2 (Fuc),
  • NANA 2 Gal 2 GlcNAc 2 (Fuci- 2 )Man 3 GlcNAc 2 or in an al, 2-linkage fucose to produce a glycoform selected from the group consisting of Gal(Fuc)GlcNAc 2 Man 3 GlcNAc 2 , Gal 2 (Fuci- 2 )GlcNAc 2 Man 3 GlcNAc 2 , NANAGal 2 (Fuci-2)GlcNAc 2 Man 3 GlcNAc 2 , and NANA 2 Gal 2 (Fuci- 2 )GlcNAc 2 Man GlcNAc 2 .
  • the antibodies e.g ., humanized antibodies
  • antigen-binding fragments thereof comprise high mannose /V-glycans, including but not limited to, Man 8 GlcNAc 2 , Man 7 GlcNAc 2 , Man 6 GlcNAc 2 , MansGlcNAc 2 , Man 4 GlcNAc 2 , or N- glycans that consist of the Man3GlcNAc2 /V-glycan structure.
  • the complex /V-glycans further include fucosylated and non-fucosylated bisected and multiantennary species.
  • /V-glycan and “glycoform” are used interchangeably and refer to an /V-linked oligosaccharide, for example, one that is attached by an asparagine-/V- acetylglucos amine linkage to an asparagine residue of a polypeptide.
  • N- linked glycoproteins contain an /V-acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in the protein.
  • glycoproteins The predominant sugars found on glycoproteins are glucose, galactose, mannose, fucose, /V-acetylgalactosamine (GalNAc), N- acetylglucosamine (GlcNAc) and sialic acid (e.g., /V-acetyl-neuraminic acid (NANA)).
  • GalNAc /V-acetylgalactosamine
  • NANA NANA
  • N- glycans have a common pentasaccharide core of Man 3 GlcNAc 2 ("Man” refers to mannose; “Glc” refers to glucose; and “NAc” refers to /V-acetyl; GlcNAc refers to N- acetylglucosamine).
  • Man refers to mannose
  • Glc refers to glucose
  • NAc refers to /V-acetyl
  • GlcNAc refers to N- acetylglucosamine
  • the reducing end of the /V-glycan is the end that is attached to the Asn residue comprising the glycosylation site on the protein.
  • N- glycans differ with respect to the number of branches (antennae) comprising peripheral sugars (e.g., GlcNAc, galactose, fucose and sialic acid) that are added to the branch (antennae) comprising peripheral sugars (e.g., GlcNAc, galactose, fucose and sialic acid) that are added to the
  • Man3 GlcNAc 2 (“Man3") core structure which is also referred to as the "trimannose core", the "pentasaccharide core” or the “paucimannose core”.
  • /V-glycans are classified according to their branched constituents (e.g., high mannose, complex or hybrid).
  • a "high mannose” type /V-glycan has five or more mannose residues.
  • a "complex” type /V-glycan typically has at least one GlcNAc attached to the 1,3 mannose arm and at least one GlcNAc attached to the 1,6 mannose arm of a "trimannose" core.
  • Complex /V-glycans may also have galactose (“Gal”) or N- acetylgalactosamine (“GalNAc”) residues that are optionally modified with sialic acid or derivatives (e.g., "NANA” or “NeuAc”, where “Neu” refers to neuraminic acid and “Ac” refers to acetyl).
  • Gal galactose
  • GalNAc N- acetylgalactosamine residues
  • sialic acid or derivatives e.g., "NANA” or “NeuAc”, where “Neu” refers to neuraminic acid and “Ac” refers to acetyl.
  • Complex /V-glycans may also have intrachain substitutions comprising "bisecting" GlcNAc and core fucose (“Fuc").
  • Complex /V-glycans may also have multiple antennae on the "trimannose core,” often referred to as “multiple antennary glycans.”
  • a "hybrid" N-glycan has at least one GlcNAc on the terminal of the 1,3 mannose arm of the trimannose core and zero or more mannoses on the 1,6 mannose arm of the trimannose core.
  • the various N-glycans are also referred to as "glycoforms".
  • G-2 refers to an /V-glycan structure that can be characterized as Man3GlcNAc2
  • G-l refers to an /V-glycan structure that can be characterized as GlcNAcMan3GlcNAc2
  • GO refers to an /V-glycan structure that can be characterized as GlcNAc2Man3GlcNAc2
  • Gl refers to an /V-glycan structure that can be characterized as GalGlcNAc2Man3GlcNAc2
  • G2 refers to an /V-glycan structure that can be characterized as GalGlcNAc2Man3GlcNAc2
  • G2 refers to an /V-glycan structure that can be characterized as
  • Gal2GlcNAc2Man3GlcNAc2 refers to an /V-glycan structure that can be characterized as NANAGal2GlcNAc2Man3GlcNAc2; and, the term “A2” refers to an N- glycan structure that can be characterized as NANA2Gal2GlcNAc2Man3GlcNAc2 ⁇
  • G-2 refers to /V-glycan species that lack fucose attached to the GlcNAc residue at the reducing end of the /V-glycan.
  • F indicates that the /V-glycan species contains a fucose residue on the GlcNAc residue at the reducing end of the N- glycan.
  • GOF, G1F, G2F, A1F, and A2F all indicate that the /V-glycan
  • multiantennary /V-glycan refers to /V-glycans that further comprise a GlcNAc residue on the mannose residue comprising the non reducing end of the 1,6 arm or the 1,3 arm of the /V-glycan or a GlcNAc residue on each of the mannose residues comprising the non-reducing end of the 1,6 arm and the 1,3 arm of the N- glycan.
  • multiantennary /V-glycans can be characterized by the formulas GlcNAc(2- 4)Man3GlcNAc2, Gal(i_4)GlcNAc(2-4)Man3GlcNAc2, or NANA( ] _4jGal( ] _4jGlcNAc(2- 4 Man GlcNAc2 ⁇
  • the term " 1-4" refers to 1, 2, 3, or 4 residues.
  • bisected /V-glycan refers to /V-glycans in which a GlcNAc residue is linked to the mannose residue at the reducing end of the N- glycan.
  • a bisected /V-glycan can be characterized by the formula GlcNAc3Man3GlcNAc2 wherein each mannose residue is linked at its non-reducing end to a GlcNAc residue.
  • GlcNAc3Man3GlcNAc2 a multiantennary /V-glycan is characterized as GlcNAc3Man3GlcNAc2
  • the formula indicates that two GlcNAc residues are linked to the mannose residue at the non- reducing end of one of the two arms of the /V-glycans and one GlcNAc residue is linked to the mannose residue at the non-reducing end of the other arm of the /V-glycan.
  • the antibodies and antigen-binding fragments thereof disclosed herein may further contain one or more glycosylation sites in either the light or heavy chain immunoglobulin variable region. Such glycosylation sites may result in increased immunogenicity of the antibody or fragment or an alteration of the pK of the antibody due to altered antigen-binding (Marshall et al. (1972)
  • Each antibody or antigen-binding fragment e.g ., 20E6 or humanized versions thereof
  • the pi for an IgGl antibody typically falls within the pH range of 7-9.5 and the pi for an IgG4 antibody typically falls within the pH range of 6-8.
  • Each antibody or antigen-binding fragment (e.g., 20E6 or humanized versions thereof) will have a characteristic melting temperature, with a higher melting temperature indicating greater overall stability in vivo (Krishnamurthy R and Manning MC (2002) Curr Pharm
  • the TMI (the temperature of initial unfolding) may be greater than 60°C, greater than 65°C, or greater than 70°C.
  • the melting point of an antibody or fragment can be measured using differential scanning calorimetry (Chen etal (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52) or circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9).
  • antibodies and antigen-binding fragments thereof e.g., antibody 20E6 and humanized versions thereof
  • Degradation of an antibody or fragment can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander AJ and Hughes DE (1995) Anal Chem 67:3626-32).
  • antibodies e.g., antibody 20E6 and humanized versions thereof
  • antigen-binding fragments thereof are selected that have minimal aggregation effects, which can lead to the triggering of an unwanted immune response and/or altered or unfavorable pharmacokinetic properties.
  • antibodies and fragments are selected that have minimal aggregation effects, which can lead to the triggering of an unwanted immune response and/or altered or unfavorable pharmacokinetic properties.
  • antibodies and fragments are selected that have minimal aggregation effects, which can lead to the triggering of an unwanted immune response and/or altered or unfavorable pharmacokinetic properties.
  • antibodies and fragments are selected that have minimal aggregation effects, which can lead to the triggering of an unwanted immune response and/or altered or unfavorable pharmacokinetic properties.
  • Aggregation can be measured by several techniques, including size-exclusion column (SEC), high performance liquid chromatography (HPLC), and light scattering.
  • SEC size-exclusion column
  • HPLC high performance liquid chromatography
  • Multispecific antibodies e.g., bispecific antibodies
  • Multispecific antibodies include at least one binding region for a particular epitope on LAP-TGFpi (e.g., human LAP-TGFpi ) as described herein, and at least one other binding region (e.g ., a cancer antigen).
  • Multispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab') 2 antibodies).
  • multispecific antibodies are well known in the art (see, e.g., PCT Publication numbers WO 05117973 and WO 06091209).
  • production of full length multispecific antibodies can be based on the co-expression of two paired immunoglobulin heavy chain-light chains, where the two chains have different specificities.
  • Various techniques for making and isolating multispecific antibody fragments directly from recombinant cell culture have also been described.
  • multispecific antibodies can be produced using leucine zippers.
  • Another strategy for making multispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported.
  • Suitable multispecific molecule platforms include, but are not limited to, Dual Targeting (DT)-Ig (GSK/Domantis), Two-in-one Antibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center), Fcab and mAb 2 (F-Star), CovX-body (CovX/Pfizer), Dual Variable Domain (DVD)-Ig (Abbott), IgG-like Bispecific (ImClone/Eli Lilly), Ts2Ab
  • the multispecific antibody comprises a first antibody (or binding portion thereof) which binds to LAP-TGEP 1 derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a multispecific molecule that binds to LAP-TGEpi and a non-LAP target molecule.
  • another functional molecule e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a multispecific molecule that binds to LAP-TGEpi and a non-LAP target molecule.
  • An antibody may be derivatized or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and/or target molecules.
  • an antibody disclosed herein can be functionally linked ( e.g ., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, receptor, or binding mimetic, such that a multispecific molecule results.
  • multispecific molecules for example, bispecific antibodies and bifunctional antibodies, comprising at least one first binding specificity for a particular epitope on LAP- TGFp 1 (e.g., human LAP-TGFp 1 ) and a second binding specificity for a second target are contemplated.
  • the second target is the second binding region specifically binds to a tumor-associated antigen. Tumor-associated antigens are well known in the art.
  • Exemplary tumor-associated antigens include, but are not limited to, AFP, ALK, BAGE proteins, b-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CCR5, CD19, CD20, CD30, CDK4, CEA, cyclin-Bl, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-l, FOLR1, GAGE proteins (e.g., GAGE-l, -2), GD2, GD3, GloboH, glypican-3, GM3, gplOO, Her2, HLA/B-raf, HLA/k-ras, HLA/M AGE- A3 , hTERT, LMP2, MAGE proteins (e.g., MAGE-l, -2, -3, -4, -6, and -12), MART-
  • the second binding region of the bispecific antibody specifically binds to CD4, CD8, CD45, CD56, CD14, CD16, CD19, CD20, CD25, CD38, CDl lb, CD22, CD30, CD39, CD114, CD23, CD73, CD163, CD206, CD203, CD200R, PD-l, PD-L1, PD-L2, CTLA-4, IDO, TIM-3, LAG-3, TIGIT, PVR, PVRL2, B7H3, B7H4, CSF-1R, VISTA, KIR, OX- 40, GITR, 4-1BB, CD40, CD40L, CD27/CD70, CD28, ICOS, CD3, CD56, NKG2DA,
  • the second binding region has agonistic properties when binding to a target, e.g., a TNF family member agonist, 0X40 ligand, CD137 ligand, CD137 agonist, STING agonist, GITR agonist, ICOS agonist, and CD28 agonist.
  • a target e.g., a TNF family member agonist, 0X40 ligand, CD137 ligand, CD137 agonist, STING agonist, GITR agonist, ICOS agonist, and CD28 agonist.
  • the antibody is a trispecific antibody comprising a first, second, and third binding region, wherein the first binding region comprises the binding specificity (e.g., antigen-binding region) of an anti-LAP antibody described herein, and the second and third binding regions bind to two different targets (or different epitopes on the same target), for example, the targets described above.
  • first binding region comprises the binding specificity (e.g., antigen-binding region) of an anti-LAP antibody described herein
  • the second and third binding regions bind to two different targets (or different epitopes on the same target), for example, the targets described above.
  • the antibody is a bifunctional antibody comprising an anti-LAP antibody described herein and a receptor molecule (i.e ., a receptor trap construct such as a TGFP superfamily ligand receptor (e.g ., ActRIIB and variants thereof) or VEGFR).
  • a receptor molecule i.e ., a receptor trap construct such as a TGFP superfamily ligand receptor (e.g ., ActRIIB and variants thereof) or VEGFR.
  • the multispecific molecules comprise as a binding specificity at least one antibody, or an antibody fragment thereof, including, e.g., an Fab, Fab', F(ab')2, Fv, or a single chain Fv.
  • the antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a single chain construct as described in Ladner et al. U.S. Patent No. 4,946,778.
  • the multispecific molecules can be prepared by conjugating the constituent binding specificities, e.g., the anti-FcR and anti-LAP binding specificities, using methods known in the art. For example, each binding specificity of the multispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation.
  • cross-linking agents examples include protein A, carbodiimide, N-succinimidyl-S-acetyl- thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N- maleimidomethyl) cyclohaxane-l-carboxylate (sulfo-SMCC).
  • Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).
  • the binding specificities are antibodies, they can be conjugated via sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains.
  • the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.
  • both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell.
  • This method is particularly useful where the multispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')2 or ligand x Fab fusion protein.
  • a multispecific molecule can be a single chain molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding
  • Multispecific molecules may comprise at least two single chain molecules. Methods for preparing multispecific molecules are described for example in U.S. Patent Number 5,260,203; U.S. Patent Number 5,455,030; U.S. Patent Number 4,881,175; U.S. Patent Number 5,132,405; U.S. Patent Number 5,091,513; U.S. Patent Number 5,476,786; U.S. Patent Number 5,013,653; U.S. Patent Number 5,258,498; and U.S. Patent Number 5,482,858.
  • Binding of the multispecific molecules to their specific targets can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g ., growth inhibition), or western blot assay.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS analysis bioassay (e.g ., growth inhibition), or western blot assay.
  • bioassay e.g ., growth inhibition
  • western blot assay Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.
  • the FcR-antibody complexes can be detected using e.g., an enzyme-linked antibody or antibody fragment which recognizes and specifically binds to the antibody-FcR complexes.
  • the complexes can be detected using any of a variety of other immunoassays.
  • the antibody can be radioactively labeled and used in a radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • the radioactive isotope can be detected by such means as the use of a a g-b counter or a scintillation counter or by
  • Immunoconjugates comprising the anti-LAP antibodies or antigen binding fragments thereof described herein can be formed by conjugating the antibodies to another therapeutic agent to form, e.g., an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • Suitable agents include, for example, a cytotoxic agent (e.g., a chemotherapeutic agent), a toxin (e.g. an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), and/or a radioactive isotope (i.e., a radioconjugate).
  • Additional suitable agents include, e.g., antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and anti-mitotic agents.
  • ADCs with the anti-LAP antibodies or antigen binding fragment thereof described herein e.g., conjugated to a cytotoxic agent
  • immunosuppressive cells e.g., regulatory T cells
  • Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, neomycin, and the tricothecenes.
  • diphtheria A chain nonbinding active fragments of diphtheria toxin
  • exotoxin A chain from Pseudomonas aeruginosa
  • ricin A chain abrin A chain
  • modeccin A chain alpha
  • cytotoxins or cytotoxic agents include, e.g., taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.
  • the antibody and therapeutic agent preferably are conjugated via a cleavable linker such as a peptidyl, disulfide, or hydrazone linker. More preferably, the linker is a peptidyl linker such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ ID NO: 214), Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu.
  • the ADCs can be prepared as described in U.S. Pat. Nos.
  • radionuclides are available for the production of radioconjugated anti-LAP antibodies. Examples include 212 Bi, 131 1, 131 In, 90 Y and 186 Re.
  • Immunoconjugates can also be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity (e.g ., lymphokines, tumor necrosis factor, IFNy, growth factors).
  • Immunoconjugates can be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2, 4-dinitrobenzene).
  • SPDP N-succinimidyl-3-(2-pyri
  • Carbon- l4-labeled 1- isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid is an exemplary chelating agent for conjugation of radionucleotide to the antibody (see, e.g., PCT publication number W094/11026).
  • anti-LAP antibodies or antigen binding fragments described herein also are used for diagnostic purposes. Such antibodies or antigen binding fragments can be conjugated to an appropriate detectable agent to form an immunoconjugate.
  • appropriate agents are detectable labels that include radioisotopes, for whole body imaging, and
  • radioisotopes enzymes, fluorescent labels and other suitable antibody tags for sample testing.
  • the detectable labels can be any of the various types used currently in the field of in vitro diagnostics, including particulate labels, isotopes, chromophores, fluorescent markers, luminescent markers, metal labels (e.g., for CyTOF, imaging mass cytometry), phosphorescent markers and the like, as well as enzyme labels that convert a given substrate to a detectable marker, and polynucleotide tags that are revealed following amplification such as by polymerase chain reaction.
  • Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase and the like.
  • the label can be the enzyme alkaline phosphatase, detected by measuring the presence or formation of chemiluminescence following conversion of 1,2 dioxetane substrates such as adamantyl methoxy phosphoryloxy phenyl dioxetane (AMPPD), disodium 3- (4-(methoxyspiro ⁇ l,2-dioxetane-3,2'-(5'-chloro)tricyclo ⁇ 3.3.l.l 3,7 ⁇ decan ⁇ -4-yl) phenyl phosphate (CSPD), as well as CDP and CDP-star® or other luminescent substrates well-known to those in the art, for example the chelates of suitable lanthanides such as Terbium(III) and Europium(III).
  • the detection means is determined by the chosen label. Appearance of the label or its reaction products can be achieved using the naked eye, in the case where the label is particulate and accumulates at appropriate levels, or using instruments such as a
  • spectrophotometer a luminometer, a fluorimeter, and the like, all in accordance with standard practice.
  • conjugation methods result in linkages which are substantially (or nearly) non-immunogenic, e.g., peptide- (i.e. amide-), sulfide-, (sterically hindered), disulfide-, hydrazone-, and ether linkages.
  • linkages are nearly non-immunogenic and show reasonable stability within serum (see e.g. Senter, P. D., Curr. Opin. Chem. Biol. 13 (2009) 235- 244; and PCT Publication numbers WO 2009/059278 and WO 95/17886).
  • coupling to the C-terminal end of the antibody or moiety is performed.
  • C-terminal modification of a protein e.g. of a Fab-fragment can e.g. be performed as described (Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371).
  • site specific reaction and covalent coupling is based on transforming a natural amino acid into an amino acid with a reactivity which is orthogonal to the reactivity of the other functional groups present.
  • a specific cysteine within a rare sequence context can be enzymatically converted in an aldehyde (see Frese, M.
  • Site specific reaction and covalent coupling can also be achieved by the selective reaction of terminal amino acids with appropriate modifying reagents.
  • the reactivity of an N-terminal cysteine with benzonitrils can be used to achieve a site- specific covalent coupling.
  • Native chemical ligation can also rely on C-terminal cysteine residues (Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).
  • the moiety may also be a synthetic peptide or peptide mimic.
  • a polypeptide is chemically synthesized, amino acids with orthogonal chemical reactivity can be incorporated during such synthesis (see e.g. de Graaf, A. J. et al., Bioconjug. Chem. 20 (2009) 1281-1295). Since a great variety of orthogonal functional groups is at stake and can be introduced into a synthetic peptide, conjugation of such peptide to a linker is standard chemistry.
  • the moiety attached to an anti-LAP antibody or antigen binding fragment is selected from the group consisting of a detectable moiety, binding moiety, a labeling moiety, and a biologically active moiety.
  • anti-LAP antibodies or antigen binding fragments disclosed herein can be tested for desired properties, e.g., those described herein, using a variety of assays known in the art.
  • the antibodies are or antigen binding fragments tested for specific binding to LAP-TGEP 1 (e.g., human LAP-TGEP 1 ).
  • LAP-TGEP 1 e.g., human LAP-TGEP 1
  • Methods for analyzing binding affinity, cross-reactivity, and binding kinetics of various anti-LAP antibodies or antigen binding fragments include standard assays known in the art, for example, BiacoreTM surface plasmon resonance (SPR) analysis using a BiacoreTM 2000 SPR instrument (Biacore AB, Uppsala, Sweden) or bio-layer interferometry (e.g ., ForteBio assay), as described in the Examples.
  • the LAP used in the binding assay is complexed with TGFpi .
  • the LAP used in the binding assay is not complexed with TGFp 1. In some embodiments, the LAP used in the binding assay is complexed with TGFpi and GARP or a fragment of GARP or LRRC33 or a fragment of LRRC33. In some embodiments the LAP used in the binding assay is complexed with TGFpi and LTBP (e.g., LTBP1, LTBP3, or LTBP4) or a fragment of LTBP.
  • LTBP e.g., LTBP1, LTBP3, or LTBP4
  • the antibodies or antigen binding fragments are tested for the ability to bind to cells that have been transfected with LAP-TGFp 1. In some embodiments the cells have also been transfected with GARP or LRRC33.
  • the antibodies or antigen binding fragments are screened for the ability to bind to the surface of beads that have been coated with LAP.
  • the antibodies or antigen binding fragments are screened for the ability to bind to LAP on cells expressing a heparin sulfate glycoprotein such as syndecan-4.
  • heparin sulfate glycoprotein-expressing cells are incubated with LAP or with LAP complexed to LTBP (e.g., LTBP1, LTBP3, or LTBP4) and the antibodies are screened for binding by flow cytometry.
  • LTBP e.g., LTBP1, LTBP3, or LTBP4
  • the antibodies or antigen binding fragments are tested for the ability to bind or affect TGFp 1. In one embodiment, the antibodies are screened for the ability to bind or affect TGFp2. In one embodiment, the antibodies are tested for the ability to bind or affect TGFp3.
  • the antibodies or antigen binding fragments are tested for their effects on TGFP activation (e.g., inhibition, stimulation, or no effect).
  • TGFp 1 activation is mediated by the binding of integrins including, but not limited, to anb ⁇ , anb8, anb3, or anb ⁇ .
  • TORb 1 activation is mediated by matrix
  • metalloproteases including, but not limited to, MMP2 and MMP9.
  • MMP2 metalloproteases
  • MMP9 metalloproteases
  • T ⁇ RbI activation is mediated by thrombospondin.
  • TORbI activation is mediated by serum proteases.
  • T ⁇ EbI activation is mediated by heat, by shear forces, by a shift in pH or by ionizing radiation.
  • T ⁇ EbI activation is mediated by reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • the source of LAP in the activation assays can be LAP on the surface of a transfected cell line, LAP on the surface of a cell population that expresses LAP endogenously or in response to specific stimuli, LAP bound to extracellular matrix, LAP in solution (e.g ., recombinant LAP), either complexed with T ⁇ Eb 1 or without TGFp 1 or complexed with T ⁇ Eb 1 and an anchor protein, such as GARP, LRRC33, LTBP1, LTBP3, or LTBP4.
  • LAP-TGFpl can be purchased from R&D Systems or can be isolated from cell supernatants.
  • the effect an antibody has on TGEpi activation can be determined, for example, using an ELISA (e.g., as described in Example 4) which measures levels of active TGFp 1 under different conditions (e.g., with or without antibody).
  • the effect an antibody has on LAP-TGFp 1 activation can also be determined using a reporter cell line that expresses TGFP receptor and responds to mature TGFp.
  • the antibodies or antigen binding fragments are tested for the ability to bind LAP in the extracellular matrix.
  • Suitable methods for determining whether antibodies bind to LAP in the extracellular matrix include in vitro assays, wherein cells (e.g., P3U 1 cells transfected with LAP-TGFP) are cultured to lay down ECM on culture plates and subsequently removed, and labeled antibodies are tested for their ability to bind to the LAP and ECM left on the culture plate surface (e.g., as described in Example 5). Similar assays can be run using fibroblast cell lines or other cells that are known to secrete LAP-TGFP and
  • whether or not the anti-LAP antibodies bind to or do not bind to ECM can be determined by an ELISA, where the ECM has been shown to express latent TGFP using commercially available antibodies.
  • the antibodies or antigen binding fragments are tested for their ability to bind to particular cell types, e.g., immune cells (e.g., immunosuppressive cells, leukocytes) or platelets.
  • immune cells e.g., immunosuppressive cells, leukocytes
  • platelets e.g
  • Antibodies or antigen binding fragments can also be tested for their ability to inhibit the proliferation or viability of cells (either in vivo or in vitro), such as tumor cells, using art- recognized methods (e.g., 3H-thymidine incorporation, immunohistochemistry with proliferation markers, animal cancer models).
  • art- recognized methods e.g., 3H-thymidine incorporation, immunohistochemistry with proliferation markers, animal cancer models.
  • Antibodies or antigen binding fragments can also be tested for their anti-tumor activity in vivo (e.g., as monotherapy or combination therapy), using syngeneic tumor models well known in the art, such as the CT26 colorectal tumor model, EMT6 breast cancer model, and 4T1 breast cancer tumor metastasis model.
  • Anti-LAP antibodies can also be tested in tumor xenogragft models which are known to be inhibited by anti-TGF antibodies (e.g., Detroit 562 tumor xenograft model). Exemplary methods for treating these models with anti-LAP antibodies are described, e.g., in Examples 12-16.
  • compositions comprising the anti-LAP antibodies or antigen binding fragments described herein, immunoconjugates comprising the same, or bispecific antibodies comprising the same, and a carrier (e.g., pharmaceutically acceptable carrier).
  • a carrier e.g., pharmaceutically acceptable carrier
  • compositions disclosed herein can include other compounds, drugs, and/or agents used for the treatment of various diseases (e.g., cancer, fibrosis, autoimmune diseases).
  • diseases e.g., cancer, fibrosis, autoimmune diseases.
  • Such compounds, drugs, and/or agents can include, for example, an anti cancer agent, a chemotherapeutic agent, an immunosuppressive agent, an immuno stimulatory agent, an immune checkpoint inhibitor, and/or an anti-inflammatory agent.
  • Exemplary compounds, drugs, and agents that can be formulated together or separately with the anti-LAP antibodies or antigen binding fragments described herein are described in the next section ( i.e ., Section IX; Uses and Methods).
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration ( e.g ., by injection or infusion).
  • the active compound i.e., antibody, immunoconjugate, or bispecific molecule
  • the active compound i.e., antibody, immunoconjugate, or bispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
  • the pharmaceutical compounds described herein may include one or more
  • a "pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like
  • nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'- dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
  • a pharmaceutical composition described herein may also include a pharmaceutically acceptable anti-oxidant.
  • pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions described herein is contemplated.
  • composition may comprise a preservative or may be devoid of a preservative.
  • Supplementary active compounds can be incorporated into the compositions.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein.
  • the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredient, preferably from about 0.1 per cent to about 70 per cent, most preferably from about 1 per cent to about 30 per cent of active ingredient in combination with a pharmaceutically acceptable carrier.
  • Dosage regimens are adjusted to provide the optimum desired response (e.g ., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the dosage unit forms described herein are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 or 10 mg/kg, of the host body weight.
  • An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months.
  • An antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease.
  • the patient can be administered a prophylactic regime.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions described herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions described herein employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • the therapeutically effective dosage of an anti-LAP antibody or antigen binding fragment in various embodiments results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
  • a therapeutically effective dose preferably results in increased survival, and/or prevention of further deterioration of physical symptoms associated with cancer.
  • a therapeutically effective dose may prevent or delay onset of cancer, such as may be desired when early or preliminary signs of the disease are present.
  • a composition described herein can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Preferred routes of administration for antibodies described herein include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion.
  • an antibody or antigen binding fragment described herein can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • a non-parenteral route such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • the active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,
  • compositions can be administered with medical devices known in the art.
  • a therapeutic composition described herein can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or
  • Examples of well-known implants and modules for use with anti-LAP antibodies described herein include: U.S. Patent No. 4,487,603, which discloses an implantable micro infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent
  • the anti-LAP antibodies or antigen binding fragments described herein can be formulated to ensure proper distribution in vivo.
  • the blood-brain barrier excludes many highly hydrophilic compounds.
  • the therapeutic compounds described herein cross the BBB (if desired, e.g., for brain cancers)
  • they can be formulated, for example, in liposomes.
  • liposomes For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,811; 5,374,548; and 5,399,331.
  • the liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade (1989) J.
  • targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016 to Low et al .); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother.
  • the antibodies, antibody compositions, and methods described herein have numerous in vitro and in vivo utilities.
  • a method of treating cancer comprising administering to a subject in need thereof an anti-LAP antibody or antigen binding fragment described herein, such that the subject is treated, e.g., such that growth of cancerous tumors is inhibited or reduced and/or that the tumors regress and/or that prolonged survival is achieved.
  • a method of treating cancer comprising administering to a subject in need thereof an effective amount (e.g ., a therapeutically effective amount) of an anti-LAP antibody described herein (or a bispecific antibody or immunoconjugate comprising the antibody).
  • the subject is administered a further therapeutic agent.
  • the further therapeutic agent is selected from the group consisting of: an anti-PD-l antibody or an antigen binding fragment thereof, an anti-LAG3 antibody or an antigen biding portion thereof, an anti- VIST A antibody or an antigen binding fragment thereof, an anti-BTLA antibody or an antigen binding fragment thereof, an anti-TIM3 antibody or an antigen binding fragment thereof, an anti-CTLA4 antibody or an antigen binding fragment thereof, an anti-HVEM antibody or an antigen binding fragment thereof, an anti-CD27 antibody or an antigen binding fragment thereof, an anti-CD 137 antibody or an antigen binding fragment thereof, an anti-OX40 antibody or an antigen binding fragment thereof, an anti-CD28 antibody or an antigen binding fragment thereof, an anti-PDLl antibody or an antigen binding fragment thereof, an anti-PDL2 antibody or an antigen binding fragment thereof, an anti-GITR antibody or an antigen binding fragment thereof, an anti-ICOS antibody or an antigen binding fragment thereof, an anti-SIRPa antibody or an antigen binding fragment thereof, an anti-SI
  • anti-PDl antibody or antigen binding fragment thereof is pembrolizumab or an antigen biding fragment thereof.
  • the heavy and light chain sequences of pembrolizumab are set forth in SEQ ID NOs: 240 and 241, respectively.
  • the further therapeutic agent is nivolumab.
  • the heavy and light chain sequences of nivolumab are set forth in comprising SEQ ID NOs: 246 and 247.
  • the cancer is characterized by abnormal TGFP activity. In some embodiments, the cancer is associated with fibrosis. In some embodiments, the cancer is associated with infiltration of CD4+ regulatory T cells. In some embodiments, the cancer is associated with infiltration of CD8+ regulatory T cells. In some embodiments, the cancer is associate with infiltration of regulatory B cells. In some embodiments, the cancer is associated with infiltration of myeloid-derived suppressor cells. In some embodiments, the cancer is associated with infiltration of tumor-associated macrophages. In some embodiments, the cancer is associated with infiltration of innate lymphoid cells. In some embodiments, the cancer is associated with infiltration of cancer-associated fibroblasts. In some embodiments, the cancer is associated with a radiation-related increase in the above cell types.
  • the cancer is associated with an increased TGFpi activation signature. In some embodiments the cancer is associated with an EMT or an EMT signature. In some embodiments the cancer is associated with a tumor exhibiting an EMT or an EMT signature and immune infiltration. In some embodiments the cancer is associated with a tumor profile of immune exclusion. In some embodiments, the cancer is associated with increased LAP expression. In some embodiments, the cancer is associated with increased GARP expression. In some embodiments, the cancer is associated with increased LRRC33 expression.
  • Cancers whose growth may be inhibited using the anti-LAP antibodies described herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer (e.g. estrogen- receptor positive breast cancer HER2-positive breast cancer; triple negative breast cancer);
  • cancer of the peritoneum cervical cancer; cholangiocarcinoma; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including
  • gastrointestinal cancer glioblastoma
  • liver cancer e.g. hepatocellular carcinoma; hepatoma
  • intra-epithelial neoplasm kidney or renal cancer
  • larynx cancer leukemia
  • lung cancer e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung
  • lymphoma including Hodgkin's and non-Hodgkin's lymphoma;
  • melanoma myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer;
  • squamous cell cancer teratocarcinoma; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; as well as other carcinomas and sarcomas; as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS -related lymphoma; and Waldenstrom's Macro globulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasts leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses
  • Additional cancers which can be treated using the anti-LAP antibodies or antigen binding fragments described herein include metastatic pancreatic cancer, metastatic adenocarcinoma of the pancreas, stomach cancer, fibrotic cancer, glioma, malignant glioma, diffuse intrinsic pontine glioma, recurrent childhood brain neoplasm renal cell carcinoma, clear-cell metastatic renal cell carcinoma, metastatic castration resistant prostate cancer, stage IV prostate cancer, metastatic melanoma, malignant melanoma, recurrent melanoma of the skin, melanoma brain metastases, malignant melanoma of head and neck, squamous cell non-small cell lung cancer, metastatic breast cancer, follicular lymphoma, advanced B-cell NHL, HL including diffuse large B-cell lymphoma (DLBCL), multiple myeloma, chronic myeloid leukemia, adult acute myeloid leukemia in remission, adult acute myeloid leukemia with
  • rhabdomyosarcoma recurrent ewing sarcoma/peripheral primitive neuroectodermal tumor, recurrent neuroblastoma, recurrent osteosarcoma, colorectal cancer, MSI positive colorectal cancer, MSI negative colorectal cancer, nasopharyngeal nonkeratinizing carcinoma, recurrent nasopharyngeal undifferentiated carcinoma, cervical adenocarcinoma, cervical adenosquamous carcinoma; cervical squamous cell carcinoma, recurrent cervical carcinoma, anal canal squamous cell carcinoma, metastatic anal canal carcinoma, recurrent anal canal carcinoma, recurrent head and neck cancer, squamous cell of head and neck, head and neck squamous cell carcinoma (HNSCC), ovarian carcinoma, colon cancer, advanced GI cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, bone neoplasms
  • Cancers may be metastatic or may be primary cancers. Cancers may be desmoplastic or non-desmoplastic. Cancers may be recurrent cancers.
  • the anti-LAP antibodies or antigen binding fragments described herein are used to treat myelodysplastic syndromes (MDS).
  • MDS are a diverse group of malignant disorders marked by bone marrow failure due to defective hematopoiesis and production of dysplastic cells.
  • TGFP is a primary driver in MDS (Geyh el al., Haematologica 2018;103:1462-71) and agents that inhibit the function of TGFP have been proposed as therapeutics (Mies et al., Curr Hematol Malig Rep 20l6;l 1:416-24).
  • MDSCs are known to be dysregulated in MDS (Chen et al., JCI 2013;123:4595-611) and agents that reduce MDSC levels in the bone marrow are potential therapeutics.
  • the anti-LAP antibodies or antigen binding fragments described herein are used to treat myelofibrosis, which is another myeloid malignancy in which TGFpi plays a central role (Mascarenhas et al., Leukemia & Lymphoma 2014;55:450-2).
  • the cancer is resistant to checkpoint inhibitor(s).
  • the cancer is intrinsically refractory or resistant (e.g., resistant to a PD-l pathway inhibitor, PD-l pathway inhibitor, or CTLA-4 pathway inhibitor).
  • the resistance or refractory state of the cancer is acquired.
  • the anti-LAP antibodies or antigen binding fragments described herein can be used in combination with checkpoint inhibitors to overcome resistance of the cancer to the checkpoint inhibitors.
  • the anti-LAP antibodies or antigen binding fragments described herein can be used to treat tumors with a mesenchymal and/or EMT signature together with checkpoint inhibitors in combination or sequentially with agents that induce a mesenchymal phenotype, such as MAPK pathway inhibitors.
  • the anti-LAP antibodies or antigen binding fragments described herein are used to enhance the viability of immune cells ex vivo, e.g., in adoptive NK cell transfer. Accordingly, in some embodiments, anti-LAP antibodies are used in combination with adoptively transferred NK cells to treat cancer. In some embodiments, the anti-LAP antibodies or antigen binding fragments described herein are used to treat tumors with MHC loss or MHC down-regulation, as monotherapy or in combination with NK activating or enhancing treatment. In some embodiments, the anti-LAP antibodies described herein are used to treat checkpoint inhibitor resistant tumors in combination with NK activating or enhancing treatment.
  • Also provided herein is a method of treating cancer associated with an increased number of circulating platelets or an increased platelet to lymphocyte ratio comprising administering to a subject in need thereof an effective amount of an antibody or antigen binding fragment which specifically binds to LAP, wherein the antibody binds to platelets but does not cause platelet aggregation or platelet degranulation.
  • the ability of a compound to inhibit cancer can be evaluated in an animal model system predictive of efficacy in human tumors.
  • this property of a composition can be evaluated by examining the ability of the compound to inhibit using in vitro assays known to the skilled practitioner.
  • a therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject.
  • One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
  • the anti-LAP antibodies or antigen binding fragments described herein can also be used to purify LAP-TGFP 1 via immunoaffinity purification.
  • provided herein is a method of diagnosing a cancer associated with regulatory T cell infiltration comprising contacting a biological sample from a patient afflicted with the cancer with an anti-LAP antibody or antigen binding fragment described herein which binds to regulatory T cells, wherein positive staining with the antibody indicates the cancer is associated with regulatory T cell infiltration.
  • a method of diagnosing a cancer associated with GARP-negative suppressive cells comprising contacting a biological sample from a patient afflicted with the cancer with an anti-LAP antibody or antigen binding fragment described herein which binds to GARP-negative suppressive cells, wherein positive staining with the antibody and negative staining with an anti-GARP antibody indicates the cancer is associated with GARP- negative suppressive cells.
  • provided herein is a method of selecting a patient afflicted with cancer for treatment with an anti-LAP antibody or antigen binding fragment described herein, comprising contacting a biological sample from the patient with the antibody, wherein positive staining with the antibody indicates the cancer is amenable to treatment with the antibody.
  • provided herein is a method of determining the response of a patient afflicted with cancer to treatment with an anti-LAP antibody or antigen binding fragment described herein comprising contacting a biological sample from the patient with the antibody, wherein reduced staining with the antibody indicates the cancer is responding to treatment with the antibody.
  • a method of determining whether a cancer in a patient has metastasized comprising (a) identifying a patient having a cancer, (b) administering a labeled (e.g ., radiolabeled) anti-LAP antibody or antigen binding fragment described herein to the patient and determining the biodistribution of the labeled anti-LAP antibody, and (c) periodically repeating step (b) to determine whether the biodistribution of the labeled anti-LAP antibody has changed, wherein a change in the biodistribution of the labeled anti-LAP antibody is indicative that the cancer has metastasized.
  • a labeled e.g ., radiolabeled
  • fibrosis is associated with cancer.
  • the fibrosis is associated with increased levels of myeloid-derived suppressor cells (e.g. Fernandez et al., Eur Respir J 2016;48:1171-83).
  • Exemplary fibrotic disorders which can be treated with any of the anti-LAP antibodies or antigen binding fragment described herein include, but are not limited to, heart fibrosis, muscle fibrosis, skin fibrosis, liver fibrosis, soft tissue (e.g ., mediastinum or retroperitoneum) fibrosis, renal fibrosis, bone marrow fibrosis, intestinal fibrosis, joint (e.g., knee, shoulder or other joints) fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, pipestem fibrosis, nephrogenic systemic fibrosis, Crohn's disease, keloid, old
  • myocardial infarction myocardial infarction, scleroderma/systemic sclerosis, subepithelial fibrosis, arthrofibrosis, some forms of adhesive capsulitis, proliferative fibrosis, viral hepatitis induced fibrosis, drug-induced fibrosis, radiation-induced fibrosis, and fibrosis associated with cancer.
  • Also provided herein is a method of reducing the number of immunosuppressive cells in a patient before, during, or after transplantation comprising administering an effective amount of any of the anti-LAP antibodies or antigen binding fragments described herein to a patient before undergoing transplantation, during transplantation, and/or after transplantation.
  • the anti-LAP antibodies or antigen binding fragments improve graft survival.
  • T ⁇ Rb has been shown to restore regenerative failure by reducing senescence and enhancing liver regeneration, in a model of acute liver disease (acetaminophen injury mouse model) (Bird et al., Sci Transl Med 20l8;l0:eaanl230).
  • acetaminophen injury mouse model acetaminophen injury mouse model
  • a method of increasing the regenerative response in acute organ injury comprising administering to a subject with acute organ injury an effective amount of the anti- LAP antibodies or antigen binding fragments described herein.
  • T ⁇ Rb Aberrant activation of T ⁇ Rb has been shown to initiate the onset of temporomandibular joint osteoarthritis (Zheng et al., Bone Res 20l8;6:26). Accordingly, also provided herein is a method of treating a patient with temporomandibular joint osteoarthritis comprising
  • LAP-TGPpl has also been shown to mediate the differentiation of CD4+ effector cells into productively and latently infected central memory T cells during HIV-l infection (Cheung et al., J Viol 20l8;92:e0l5l0-l7). Accordingly, also provided herein is a method of treating a patient with HIV-l infection (or a patient at risk of developing HIV-l infection) comprising administering to the patient an effective amount of the anti-LAP antibodies or antigen binding fragments described herein to treat the HIV-l infection (e.g ., inhibit differentiation of CD4+ effector cells into productively and latently infected central memory T cells).
  • TGFP-expressing macrophages and suppressive regulatory T cells have been shown to be altered in the peritoneal fluid of patients with endometriosis (Hanada et al., Reprod Biol Endocrinol 20l8;l6:9), suggesting that targeting LAP-TGFbl expressed on these cells may be beneficial for treating the disorder.
  • a method of treating a patient with endometriosis comprising administering to the patient an effective amount of the anti-LAP antibodies or antigen binding fragments described herein to treat the endometriosis.
  • LAP-TGFp 1 -expressing CD4+ T cells and CD 14+ monocytes and macrophages have been shown to be increased in patients carrying multidrug resistant Mycobacterium tuberculosis (Basile et al., Clin Exp Immunol 2016;187: 160), suggesting that targeting LAP-TGFpi expressed on these cells may be beneficial for treating the infection.
  • a method of treating a patient with multidrug resistant Mycobacterium tuberculosis comprising administering to the patient an effective amount of the anti-LAP antibodies or antigen binding fragments described herein (e.g., anti-LAP antibodies which inhibit LAP-TGEP 1 activation) to treat the infection.
  • the anti-LAP antibodies or antigen binding fragments described herein are used to treat b-thalassemia, a disorder in which TGFP superfamily members have been implicated in defective erythropoiesis (Dussiot et al. Nat Med 2014;20:398-407).
  • the anti-LAP antibody or antigen binding fragment can be used as monotherapy to treat a disease or disorder (e.g., cancer).
  • a disease or disorder e.g., cancer
  • an anti-LAP antibody or antigen binding fragment can be used in conjunction with another agent or therapy, e.g., an anti-cancer agent, a chemotherapeutic agent, an immunosuppressive agent, an
  • immuno stimulatory agent an immune checkpoint inhibitor, an anti-inflammatory agent, or a cell therapy, as described in more detail below.
  • anti-LAP antibodies or antigen binding fragments described herein can be used in combination with various treatments or agents (or in the context of a multispecific antibody or bifunctional partner) known in the art for the treatment of cancer, as described below.
  • Suitable anti-cancer agents for use in combination therapy with the anti-LAP antibodies or antigen binding fragments described herein include, but are not limited to, surgery, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, radiotherapy and agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, and other agents to treat cancer, such as anti-HER-2 antibodies (e.g.
  • HERCEPTIN® anti-CD20 antibodies
  • an epidermal growth factor receptor (EGFR) antagonist e.g., a tyrosine kinase inhibitor
  • HER1/EGFR inhibitor e.g., erlotinib (TARCEVA®)
  • platelet derived growth factor inhibitors e.g., GLEEVEC (Imatinib Mesylate)
  • a COX-2 inhibitor e.g., celecoxib
  • interferons cytokines
  • antagonists e.g., neutralizing antibodies
  • PD 1, PDL1, PDL2 e.g., pembrolizumab; nivolumab; MK-3475; AMP-224; MPDL3280A; MEDI0680; MSB0010718C; and/or MEDI4736
  • CTLA4 e.g., tremelimumab (PFIZER) and ipilimumab
  • LAG3 e.g.,
  • Suitable chemotherapeutic agents for use in combination therapy with the anti-LAP antibodies or antigen binding fragments described herein include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; temozolomide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and
  • cryptophycin 1 and cryptophycin 8 dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g ., calicheamicin, especially calicheamicin gammall and calicheamicin omegal 1 (see, e.
  • bisphosphonates such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5- oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpho lino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mito
  • cyclophosphamide thiotepa
  • taxoids e.g., TAXOL® paclitaxel (Bristol-Myers Squibb
  • TAXOTERE® doxetaxel (Rhone- Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin, vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; NAVELBINE, vinorelbine; novantrone; teniposide; edatrexate; daunomycin;
  • aminopterin xeloda
  • ibandronate irinotecan (Camptosar, CPT-l l) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000;
  • DMFO difluoromethylornithine
  • retinoids such as retinoic acid
  • capecitabine combretastatin
  • leucovorin LV
  • oxaliplatin including the oxaliplatin treatment regimen (FOLFOX); lapatinib (TYKERB); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (TARCEVA®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • FOLFOX oxaliplatin treatment regimen
  • TYKERB lapatinib
  • inhibitors of PKC-alpha, Raf, H-Ras, EGFR e.g., erlotinib (TARCEVA®)
  • VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • drugs targeting epigenetic regulators such as HD AC inhibitors, bromodomain inhibitors, and E3 ligase (e.g., cereblon) inhibitors (e.g., lenalidomide,
  • Suitable anti-inflammatory agents for use in combination therapy with the anti-LAP antibodies or antigen binding fragments described herein include, but are not limited to, aspirin and other salicylates, Cox-2 inhibitors (e.g., rofecoxib and celecoxib), NSAIDs (such as ibuprofen, fenoprofen, naproxen, sulindac, diclofenac, piroxicam, ketoprofen, diflunisal, nabumetone, etodolac, oxaprozin, and indomethacin), anti-IL6R antibodies, anti-IL8 antibodies, anti-ILl5 antibodies, anti-ILl5R antibodies, anti-CD4 antibodies, anti-CDl la antibodies (e.g., efalizumab), anti-alpha-4/beta-l integrin (VLA4) antibodies (e.g., natalizumab), CTLA4-Ig for the treatment of inflammatory diseases, predn
  • DMARDs antirheumatic drugs
  • DMARDs such as methotrexate, hydroxychloroquine, sulfasalazine, pyrimidine synthesis inhibitors (e.g., lefhmomide), IL-l receptor blocking agents (e.g., anakinra), TNF-a blocking agents (e.g., etanercept, infliximab, and adalimumab), and the like.
  • Suitable immunomodulatory agents include, but are not limited to, cyclosporine, azathioprine, mycophenolic acid, mycophenolate mofetil, corticosteroids such as prednisone, methotrexate, gold salts,
  • sulfasalazine antimalarials, brequinar, lefhmomide, mizoribine, l5-deoxyspergualine, 6- mercaptopurine, cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, anti-thymocyte globulin, thymopentin, thymosin-a, antibodies that bind to p75 of the IL-2 receptor, antibodies that bind to MHC, CD2, CD3, CD4, CD7, CD28, B7, CD40, CD45, IFN-g, TNF-a, IL-4, IL-5, IL-6R, IL-6, IGF, IGFR1, IL-7, IL-8, IL-10, CDl la, or CD58, or antibodies binding to their ligands, soluble IL-15R, IL-10, B7 molecules (B7-1, B7-2, variants thereof, and fragments thereof), ICOS, 0
  • Additional immunosuppressive agents include, for example, anti-TNF agents such as etanercept, adalimumab and infliximab, and steroids.
  • anti-TNF agents such as etanercept, adalimumab and infliximab
  • steroids include, for example: aldosterone, beclomethasone, betamethasone, budesonide, cloprednol, cortisone, cortivazol, deoxycortone, desonide, desoximetasone, dexamethasone, difluorocortolone, fluclorolone, flumethasone, flunisolide, fluocinolone, fluocinonide, fluocortin butyl, fluorocortisone, fluorocortolone, fluorometholone, flurandrenolone, fluticasone, halcinonide, hydrocortisone, icomethasone, meprednisone,
  • Suitable immunostimulatory agents for use in combination therapy with the anti-LAP antibodies or antigen binding fragments described herein include, for example, compounds capable of stimulating antigen presenting cells (APCs), such as dendritic cells (DCs) and macrophages.
  • APCs antigen presenting cells
  • DCs dendritic cells
  • suitable immunostimulatory agents are capable of stimulating APCs, so that the maturation process of the APCs is accelerated, the proliferation of APCs is increased, and/or the recruitment or release of co-stimulatory molecules (e.g ., CD80, CD86, ICAM-l, MHC molecules and CCR7) and pro-inflammatory cytokines (e.g., IL- 1 b, IL-6, IL-12, IL-15, and IFN-g) is upregulated.
  • co-stimulatory molecules e.g ., CD80, CD86, ICAM-l, MHC molecules and CCR7
  • pro-inflammatory cytokines e.g.,
  • Suitable immunostimulatory agents are also capable of increasing T cell proliferation.
  • immunostimulatory agents include, but are not be limited to, CD40 ligand; FLT 3 ligand; cytokines, such as IFN-a, IFN-b, IFN-g and IL-2; colony- stimulating factors, such as G-CSF (granulocyte colony-stimulating factor) and GM-CSF (granulocyte-macrophage colony-stimulating factor); an anti-CTLA-4 antibody, anti-PDl antibody, anti-41BB antibody, or anti-OX-40 antibody; LPS (endotoxin); ssRNA; dsRNA; Bacille Calmette- Guerin (BCG); Levamisole hydrochloride; and intravenous immune globulins.
  • an immunostimulatory agent may be a Toll-like Receptor (TLR) agonist.
  • TLR Toll-like Receptor
  • the immunostimulatory agent may be a TLR3 agonist such as double- stranded inosine:cytosine polynucleotide (Poly I:C, for example available as AmpligenTM from
  • Hemispherx Bipharma PA, US or Poly IC:LC from Oncovir) or Poly A:U; a TLR4 agonist such as monophosphoryl lipid A (MPL) or RC-529 (for example as available from GSK, UK); a TLR5 agonist such as flagellin; a TLR7 or TLR8 agonist such as an imidazoquinoline TLR7 or TLR 8 agonist, for example imiquimod ( e.g ., AldaraTM) or resiquimod and related
  • imidazoquinoline agents e.g., as available from 3M Corporation
  • TLR 9 agonist such as a deoxynucleotide with unmethylated CpG motifs (“CpGs”, e.g., as available from Coley
  • the immuno stimulatory molecule is a STING agonist.
  • Such immunostimulatory agents may be administered simultaneously, separately or sequentially with the anti-LAP antibodies or antigen binding fragments described herein.
  • Suitable immune checkpoint blockers include, but are not limited to, agents (e.g., antibodies) that bind to PD-l, PD-L1, PD-L2, LAG-3, CTLA4, TIGIT, ICOS, 0X40, PVR, PVRIG, VISTA, and TIM3.
  • agents e.g., antibodies
  • Non-limiting examples of antibodies that bind to PD-l, PD-L1, and PD-L2 include pembrolizumab; nivolumab; MK-3475; MPDL32; MED 10680; MEDI4736; AMP-224; and MSB0010718C.
  • the anti-LAP antibodies or antigen binding fragments described herein may be administered to a subject with an agent that targets a member of the IgSF family to increase an immune response.
  • an anti-LAP antibody or antigen binding fragment may be administered with an agent that targets a member of the B7 family of membrane -bound ligands that includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6 or a co-stimulatory or co- inhibitory receptor binding specifically to a B7 family member.
  • a member of the B7 family of membrane -bound ligands that includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6 or a co-stimulatory or co- inhibitory receptor binding specifically to a B7 family member.
  • An anti-LAP antibody or antigen binding fragment may also be administered with an agent that targets a member of the TNF and TNFR family of molecules (ligands or receptors), such as CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137, TRAIL/ Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK,
  • ligands or receptors such as CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137, TRAIL/ Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK,
  • T cell responses can be stimulated by a combination of anti-LAP antibodies or antigen binding fragments described herein and one or more of the following agents:
  • An antagonist (inhibitor or blocking agent) of a protein that inhibits T cell activation e.g., immune checkpoint inhibitors
  • a protein that inhibits T cell activation e.g., immune checkpoint inhibitors
  • CTLA-4, PD-l, PD- Ll, PD-L2, and LAG-3 as described above, and any of the following proteins: TIM-3, Galectin 9, CEACAM-l, BTLA, CD69, Galectin-l, TIGIT, CD 113, CD155, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-l, and TIM-4; and/or
  • An agonist of a protein that stimulates T cell activation such as B7-1, B7-2,
  • CD28 4-1BB (CD137), 4-1BBL, GITR, ICOS, ICOS-L, 0X40, OX40L, CD70, CD27, CD40, DR3 and CD28H.
  • agents that modulate the above proteins and may be combined with the anti- LAP antibodies or antigen binding fragments described herein for treating cancer include:
  • YervoyTM ipilimumab or Tremelimumab (to CTLA-4), galiximab (to B7.1), BMS-936558 (to PD-l), MK-3475 (to PD-l), AMP224 (to B7DC), BMS-936559 (to B7-H1), MPDL3280A (to B7-H1), MEDI-570 (to ICOS), AMG557 (to B7H2), MGA271 (to B7H3), IMP321 (to LAG-3), BMS-663513 (to CD137), PF-05082566 (to CD137), CDX-1127 (to CD27), anti-OX40
  • anti-LAP antibodies or antigen binding fragments for the treatment of cancer include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells.
  • anti-LAP antibodies or antigen binding fragments can be combined with antagonists of KIR (e.g., lirilumab).
  • T cell activation is also regulated by soluble cytokines, and anti-LAP antibodies may be administered to a subject, e.g., having cancer, with antagonists of cytokines that inhibit T cell activation or agonists of cytokines that stimulate T cell activation.
  • anti-LAP antibodies or antigen binding fragments can be used in combination with (i) antagonists (or inhibitors or blocking agents) of proteins of the IgSF family or B7 family or the TNF family that inhibit T cell activation or antagonists of cytokines that inhibit T cell activation (e.g., IL-6, IL-10, TGF-B, VEGF;“immunosuppressive cytokines”) and/or (ii) agonists of stimulatory receptors of the IgSF family, B7 family or the TNF family or of cytokines that stimulate T cell activation, for stimulating an immune response, e.g., for treating proliferative diseases, such as cancer.
  • cytokines that inhibit T cell activation e.g., IL-6, IL-10, TGF-B, VEGF;“immunosuppressive cytokines”
  • agents for combination therapies include agents that inhibit or deplete macrophages or monocytes, including but not limited to CSF-1R antagonists such as CSF-1R antagonist antibodies including RG7155 (see PCT publication numbers WOl 1/70024,
  • agents that may be combined with anti-LAP antibodies or antigen binding fragments include agents that enhance tumor antigen presentation, e.g., dendritic cell vaccines, GM-CSF secreting cellular vaccines, CpG oligonucleotides, and imiquimod, or therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines).
  • agents that enhance tumor antigen presentation e.g., dendritic cell vaccines, GM-CSF secreting cellular vaccines, CpG oligonucleotides, and imiquimod
  • therapies that enhance the immunogenicity of tumor cells e.g., anthracyclines.
  • Another therapy that may be combined with anti-LAP antibodies is a therapy that inhibits a metabolic enzyme such as indoleamine dioxygenase (IDO), tryptophan-2, 3-dioxygenase, dioxygenase, arginase, or nitric oxide synthetase.
  • IDO indoleamine dioxygenase
  • tryptophan-2 tryptophan-2
  • 3-dioxygenase dioxygenase
  • dioxygenase arginase
  • nitric oxide synthetase nitric oxide synthetase
  • agents that may be used with anti-LAP antibodies includes agents that inhibit the formation of adenosine or inhibit the adenosine A2A receptor, for example, anti- CD73 antibodies, anti-CD39 antibodies, and adenosine A2A/A2b inhibitors.
  • therapies that may be combined with anti-LAP antibodies or antigen binding fragments for treating cancer include therapies that reverse/prevent T cell anergy or exhaustion and therapies that trigger an innate immune activation and/or inflammation at a tumor site.
  • the anti-LAP antibodies or antigen binding fragments may be combined with a combinatorial approach that targets multiple elements of the immune pathway, such as one or more of the following: a therapy that enhances tumor antigen presentation (e.g., dendritic cell vaccine, GM-CSF secreting cellular vaccines, CpG oligonucleotides, imiquimod); a therapy that inhibits negative immune regulation e.g., by inhibiting CTLA-4 and/or PD1/PD-L1/PD-L2 pathway and/or depleting or blocking regulatory T cells or other immune suppressing cells; a therapy that stimulates positive immune regulation, e.g., with agonists that stimulate the CD- 137 and/or GITR pathway and/or stimulate T cell effector function; a therapy that increases systemically the frequency of anti-tumor T cells; a therapy that depletes or inhibits regulatory T cells using an antagonist of CD25 (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion
  • T cell anergy or exhaustion reverses/prevents T cell anergy or exhaustion; a therapy that triggers an innate immune activation and/or inflammation at a tumor site; administration of immune stimulatory cytokines; or blocking of immunosuppressive or immunorepressive cytokines.
  • anti-LAP antibodies or antigen binding fragments described herein can be combined with proinflammatory cytokines, for example, IL-12 and IL-2. These cytokines can be modified to enhance half-life and tumor targeting.
  • anti-LAP antibodies or antigen binding fragments described herein can be combined with immune cell engagers such as NK cell engagers or T cell engagers.
  • anti-LAP antibodies or antigen binding fragments described herein can be combined with indoleamine dioxygenase (IDO) inhibitors, tryptophan-2,3 -dioxygenase (TDO) inhibitors, and dual IDO/TDO inhibitors.
  • IDO indoleamine dioxygenase
  • TDO tryptophan-2,3 -dioxygenase
  • dual IDO/TDO inhibitors dual IDO/TDO inhibitors.
  • anti-LAP antibodies or antigen binding fragments described herein can be combined with kynurine inhibitors.
  • anti-LAP antibodies or antigen binding fragments described herein can be combined with CD47 and/or SIRPa blocking therapies.
  • anti-LAP antibodies or antigen binding fragments described herein can be combined with JAK inhibitors and JAK pathway inhibitors (e.g., STAT3 inhibitors), e.g., for the treatment of myelofibrosis and myeloproliferative neoplasms.
  • JAK inhibitors and JAK pathway inhibitors e.g., STAT3 inhibitors
  • anti-LAP antibodies or antigen binding fragments described herein can be combined with DNA damage repair inhibitors.
  • anti-LAP antibodies or antigen binding fragments described herein can be combined with erythropoietin and drugs that stimulate hematopoiesis.
  • anti-LAP antibodies or antigen binding fragments described herein can be combined with angiogenesis inhibitors.
  • anti-LAP antibodies or antigen binding fragments described herein can be combined with anti- viral drugs, such as neuramidase inhibitors.
  • Bispecific antibodies which have a first binding region with the specificity of the anti- LAP antibodies or antigen binding fragments described herein and a second binding region which binds to an immune checkpoint blocker (e.g ., PD-l, PD-L1) can be used in combination with at least one additional anti-cancer agent (e.g., radiation, chemotherapeutic agents, biologies, vaccines) to inhibit tumor growth.
  • additional anti-cancer agent e.g., radiation, chemotherapeutic agents, biologies, vaccines
  • anti-LAP antibodies or antigen binding fragments described herein can be combined with one or more immuno stimulatory antibodies, such as an anti-PD-l antagonist antibody, an anti-PD-Ll antagonist antibody, an antagonist anti-CTLA-4 antibody, an antagonistic anti-TIM3 antibody, and/or an anti-LAG3 antagonist antibody, such that an immune response is stimulated in the subject, for example to inhibit tumor growth.
  • immuno stimulatory antibodies such as an anti-PD-l antagonist antibody, an anti-PD-Ll antagonist antibody, an antagonist anti-CTLA-4 antibody, an antagonistic anti-TIM3 antibody, and/or an anti-LAG3 antagonist antibody, such that an immune response is stimulated in the subject, for example to inhibit tumor growth.
  • Exemplary anti-PD-l antibodies include nivolumab, pembrolizumab(also known as MK- 3475,Lambrolizumab) described in WO2012/145493; AMP-514 described in WO 2012/145493, as well as PD-l antibodies and other PD-l inhibitors described in WO 2009/014708, WO 03/099196, WO 2009/114335, WO 2011/066389, WO 2011/161699, WO 2012/145493, U.S. Patent Nos. 7,635,757 and 8,217,149, and U.S. Patent Publication No. 2009/0317368.
  • anti-PD-Ll antibodies include MEDI4736 (also known as Anti-B7-Hl), MPDL3280A (also known as RG7446), MSB0010718C (WO2013/79174), rHigMl2B7, as well as any of the anti-PD-Ll antibodies disclosed in WO2013/173223, WO2011/066389,
  • Exemplary anti-CTLA-4 antibodies include YervoyTM (ipilimumab), tremelimumab (formerly ticilimumab, CP-675,206), or an anti-CTLA-4 antibody described in any of the following publications: WO 98/42752; WO 00/37504; U.S. Pat. No. 6,207,156; Hurwitz et al. (1998) Proc. Natl. Acad. Sci. USA 95(17):10067-10071; Camacho et al. (2004) J. Clin. Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res.
  • Exemplary anti-LAG3 antibodies include IMP731 and IMP-321, described in US Publication No. 2011/007023, and PCT publication numbers W008/132601, and WO09/44273, as well as antibodies described in U.S. Patent Publication No. US2011/0150892, and
  • Anti-LAP antibodies or antigen binding fragments can also be combined with immune- oncology agents such as CD137 (4-1BB) agonists (e.g ., an agonistic CD137 antibody such as urelumab or PF-05082566 (see PCT publication number WO 12/32433)); GITR agonists (e.g., an agonistic anti-GITR antibody), CD40 agonists (e.g., an agonistic CD40 antibody); CD40 antagonists (e.g., an antagonistic CD40 antibody such as lucatumumab (HCD122), dacetuzumab (SGN-40), CP-870,893 or Chi Lob 7/4); CD27 agonists (e.g., an agonistic CD27 antibody such as varlilumab (CDX-1127)), MGA271 (to B7H3) (WOl 1/109400)); KIR antagonists (e.g., li), a agonistic CD137 antibody such as varlilumab
  • TLR2/4 agonists e.g., Bacillus Calmette-Guerin
  • TLR7 agonists e.g., Hiltonol or Imiquimod
  • TLR7/8 agonists e.g.,
  • TLR9 agonists e.g., CpG7909
  • TGF-b inhibitors e.g., GC1008,
  • anti-LAP antibodies or antigen binding fragments described herein can also be combined with an immunogenic agent, such as cancerous cells, purified tumor antigens
  • tumor vaccines include peptides of melanoma antigens, such as peptides of gplOO, MAGE antigens, Trp-2, MART1 and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF (discussed further below).
  • anti-LAP antibodies or antigen binding fragments described herein can also be combined with an anti-neoplastic antibody, such as Rituxan® (rituximab), Herceptin®
  • antigen specific T cells Several experimental treatment protocols involve ex vivo activation and expansion of antigen specific T cells and adoptive transfer of these cells into recipients in order to antigen- specific T cells against tumor (Greenberg & Riddell, supra).
  • Ex vivo activation in the presence of the anti-LAP antibodies described herein with or without an additional immunostimulating therapy (e.g., an immune checkpoint blocker) can be expected to increase the frequency and activity of the adoptively transferred T cells.
  • the anti-LAP antibody or antigen binding fragment may also be administered with a standard of care treatment, or another treatment, such as radiation, surgery, or chemotherapy.
  • the anti-LAP antibody or antigen binding fragment may be combined with a vaccination protocol.
  • Many experimental strategies for vaccination against tumors have been devised (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition).
  • a vaccine is prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be most effective when the tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci U.S.A. 90: 3539-43).
  • DC Dendritic cells
  • DC's can be produced ex vivo and loaded with various protein and peptide antigens as well as tumor cell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs can also be transduced by genetic means to express these tumor antigens as well. DCs have also been fused directly to tumor cells for the purposes of immunization (Kugler et al. (2000) Nature Medicine 6:332-336). As a method of vaccination, DC immunization can be effectively combined with the anti-LAP antibodies or antigen binding fragments described herein to activate more potent anti-tumor responses.
  • the combination of therapeutic antibodies discussed herein can be administered concurrently as a single composition in a pharmaceutically acceptable carrier, or concurrently as separate compositions with each antibody in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic antibodies can be administered sequentially.
  • kits comprising the anti-LAP antibodies or antigen binding fragments, multispecific molecules, or immunoconjugates disclosed herein, optionally contained in a single vial or container, and include, e.g., instructions for use in treating or diagnosing a disease (e.g., cancer).
  • the kits may include a label indicating the intended use of the contents of the kit.
  • the term label includes any writing, marketing materials or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
  • kits may comprise the antibody, multispecific molecule, or immunoconjugate in unit dosage form, such as in a single dose vial or a single dose pre-loaded syringe.
  • the CDR sequences, variable region sequences, and full-length heavy and light chain sequences of anti-LAP antibodies 28G11, 22F9, 20E6, 17G8, and 24E3 are provided in Table 34. These antibodies were prepared in murine antibody format with an mIgG2a constant region, chimeric format with a human IgG constant region, and/or in humanized format.
  • variable region sequences of each antibody were fused to a murine IgG2a constant region.
  • the murine VH domains were fused to a codon-optimized gene for the murine IgG2a constant domains (UniProt accession #P0l863) using overlap extension PCR.
  • the murine VL domains were fused to a codon- optimized gene for the murine kappa constant domain (UniProt accession #P0l837) using overlap extension PCR.
  • the complete heavy-chain and light-chain sequences were individually TOPO-TA cloned into pcDNA3.4 for expression in ExpiCHO cells.
  • variable region sequences of the murine parental clones were fused to the human IgGl constant region sequences.
  • the murine VH domains were fused to a codon-optimized gene for the human IgGl constant domains (UniProt accession #P0l857) using overlap extension PCR.
  • the murine VL domains were fused to a codon-optimized gene for the human kappa constant domain (UniProt accession #P0l834) using overlap extension PCR.
  • the complete heavy-chain and light-chain sequences were individually TOPO-TA cloned into pcDNA3.4 for expression in ExpiCHO cells.
  • This Example describes the ability of anti-LAP antibodies 28Gl l_MgGl, 22F9_MgGl, and 20E6_hIgGl to bind to human and murine LAP-TGFpi using bio-layer interferometry.
  • the chimeric antibodies were biotinylated using EZ-Link SulfoNHS-LC-Biotin
  • a strep tavidin-functionalized tip was equilibrated in binding buffer (10 mM sodium phosphate, 150 mM sodium chloride, 1% (w/v) bovine serum albumin, 0.05% (w/v) sodium azide, pH 7.4). The tip was dipped in a 10 pg/mL solution of biotinylated, chimeric anti- LAP in binding buffer for 15 seconds to load the tip with antibody.
  • the antibody-loaded tip was then washed in binding buffer and placed in a solution containing 0-24 nM of LAP-TGFpl (either a fusion protein containing a human IgGl Fc domain fused to human LAP-TGFpl or a murine LAP-TGFpl with a C-terminal polyhistidine purification tag).
  • LAP-TGFpl either a fusion protein containing a human IgGl Fc domain fused to human LAP-TGFpl or a murine LAP-TGFpl with a C-terminal polyhistidine purification tag.
  • association phase association phase
  • dissociation phase was fit to a 1:1 binding model to determine the binding rate constants.
  • This Example describes the binding of anti-LAP antibodies to LAP-TGFp isoforms and LAP-TGFP variants.
  • anti-LAP antibodies l7G8_MgGl, 24E3_hIgGl, and 2C9_(hyb) were also tested in this experiment.
  • 4 x 10 5 each of (a) HT1080 cells, (b) HT1080 cells overexpressing human LAP-TGFP 1, (c) HT1080 cells overexpressing human LAP-TGFp2, (d) HT1080 cells overexpressing human LAP-TGFP3, (e) HT1080 cells overexpressing murine LAP-TGFP 1, (f) P3U1 cells, (g) P3U1 cells overexpressing LAP-TGFpl and GARP, and (h) P3U1 cells overexpressing LAP-TGFpi and LRRC33 were cultured in 96-well plates. The plates were centrifuged for 5 min at 1,500 rpm, liquid was removed, and cells were resuspended with 200 pL FACS buffer.
  • the plates were centrifuged again, diluted primary antibody was added to each well, and the plates were incubated on ice for 20 minutes, followed by centrifugation.
  • the cells were resuspended in 200 pL FACS buffer, centrifuged again, and resuspended in 50 pL diluted secondary antibody (Alexa647-anti-Human IgG or APC-anti-Mouse IgG).
  • the plates were incubated on ice for 20 minutes in the dark, washed twice with 200 pL FACS buffer, and cells from each well (in 200 pL FACS buffer) were read on the Attune NXT instrument.
  • the anti-LAP antibodies to bind variants of TGFpi that either prevent TGFp 1 activation by integrins (“closed” conformation) or favor release (“open” conformation), chimeric TGFpi sequences containing residues from chicken TGFpl, and the LAP-only TGFpi variant ( i.e ., human TGFp 1 variant which does not contain the mature cytokine) was tested.
  • HT1080 cells each of (a) HT1080 cells, (b) HT1080 cells overexpressing human LAP- TGFpl, (c) HT1080 cells overexpressing LAP-TGFpi with K27C and Y75C mutations, (d) HT1080 cells overexpressing LAP-TGFpi with a Y74T mutation, (e) HT1080 cells
  • Antibodies 28Gl l_hIgGl, 22F9_hIgGl, 20E6_hIgGl, l7G8_MgGl, and 24E3_hIgGl bind to the K27C/Y75C (“closed”) LAP-TGFpl variant, but not to the Y74T (“open”) LAP-TGFpi variant.
  • antibody 2C9_(hyb) binds to both the K27C/Y75C and Y74T LAP-TGFpl variants.
  • anti-LAP antibodies 28Gl l_MgGl, 22F9_MgGl, 20E6_hIgGl, l7G8_MgGl, and 24E3_hIgGl, bind to free human TGFpi (i.e., mature TGFp 1 that lacks LAP) bind to free human TGFpi (i.e., mature TGFp 1 that lacks LAP)
  • TGFpi i.e., mature TGFp 1 that lacks LAP
  • mature TGFpi 1000 pg
  • an isotype control antibody as a negative control
  • the commercially available anti-TGFp antibody ID 11 as a positive control for 10 minutes on ice.
  • the 1D11 antibody is commercially available from any number of sellers, for example Bio x Cell Inc. (West Riverside, NH).
  • 20E6_MgGl, l7G8_MgGl, and 24E3_MgGl share a related epitope. All anti-LAP antibodies tested bind to HT1080 cells overexpressing wild-type human LAP-TGFpi (HT1080-1ib1), but not to untransduced HT1080 cells. Antibodies 28G1 l_MgGl, 22F9_MgGl, 20E6_hIgGl, l7G8_MgGl, and 24E3_hIgGl did not bind to the LAP-only construct or the chimera containing chicken exon #2.2. Antibody 2C9_(hyb) binds to both the LAP-only construct, but not to the exon #2.2 chimera. This compilation of data indicates that antibodies 28G1 l_MgGl,
  • 22F9_hIgGl, 20E6_MgGl, l7G8_MgGl, and 24E3_MgGl only bind to LAP that contains the mature cytokine, and this is supported by the results shown in Figure 4. However, chimeras with chicken exons #6 and #7, which encompass the mature cytokine, were bound by
  • 20E6_MgGl, l7G8_MgGl, and 24E3_MgGl do not bind the mature cytokine directly, but rather are sensitive to conformational changes in the LAP region induced by the presence or absence of the mature cytokine.
  • 2C9 binds to all variants of LAP, including the“open” and “closed” conformation variants, as well as LAP in the presence or absence of the mature cytokine.
  • the binding epitope for 28G11 was mapped by assessing binding of cell-surface chimeric human/chicken LAP-TGFP 1 molecules using flow cytometry. The use of these binding data to determine epitopes was based on the following assumptions:
  • Anti-LAP antibodies will not bind to chicken LAP-TGFP sequence
  • an anti-LAP antibody does not bind to a chimera, at least one residue in that exon is part of the epitope, or the presence of chicken sequence in that exon causes a
  • constructs were subcloned into lentivirus and transduced into HT1080 cells.
  • GFP green fluorescent protein
  • HT1080 cells 4 x 10 5 each of (a) HT1080 cells, (b) HT1080 cells overexpressing human LAP-TGFpl, (c) HT1080 cells overexpressing chicken LAP-TGFP 1 , and (d) HT1080 cells overexpressing human/chicken chimeras #l-#7 were cultured in 96-well plates. The plates were centrifuged for 5 min at 1,500 rpm, liquid was removed, and cells were resuspended with 200 pL FACS buffer. The plates were centrifuged again, diluted primary antibody was added to each well, and the plates were incubated on ice for 20 minutes, followed by centrifugation.
  • the cells were resuspended in 200 pL FACS buffer, centrifuged again, and resuspended in 50 pL diluted secondary antibody (APC-anti-Mouse IgG).
  • the plates were incubated on ice for 20 minutes in the dark, washed twice with 200 pL FACS buffer, and cells from each well (in 200 pL FACS buffer) were read on the Attune NXT instrument.
  • the antibodies were considered to be binding if >10% of cells were GFP+/APC+ and there was visible correlation between the GFP and allophycocyanin (APC) signals (Table 5). Table 5.
  • Chimera #7 was not bound by the Rmab antibody, but was recognized by both 28G1 l_(hyb) and 2C9_(hyb), showing that this construct is expressed on the cell surface, and that the epitope for Rmab is likely in this region of the protein.
  • constructs with combinations of human and chicken sequence are able to fold into the correct structure. Accordingly, additional chimeras can be generated that could be used as immunogens to target specific epitopes of interest on LAP- TGFp 1.
  • These constructs would be the inverse of the constructs described in Example 2. That is, the majority of the sequence would be taken from chicken LAP-TGFpl, with small sections of human LAP-TGFp 1 inserted in regions containing the desired epitope.
  • Exemplary epitopes on LAP-TGFpi that can be targeted using this strategy include, for example, an epitope comprising amino acids 82-130 of human LAP-TGFpl, the lower arm of LAP-TGFpl, or the latency loop of LAP-TGEpi .
  • This chimeric protein could be used to immunize chickens to yield monoclonal antibodies. Since the chicken LAP-TGEP 1 would be recognized as self, the immune response will be focused on the human sequence. Exemplary chicken-human chimeras which can be used to immunize chickens is shown in Table 6. The sequences of these chimeras are provided in Table 34.
  • Anti-LAP antibodies generated in this manner can be tested for various functions (e.g., binding to human LAP-TGEpi , inhibition of TGFpl activation, binding to immune cells) using the methods described herein.
  • This Example describes the ability of 28Gl l_MgGl, 22F9_hIgGl, 20E6_MgGl, 2C9_mIgG2a, l6B4_mIgG2a, l7G8_MgGl, and 24E3_MgGl antibodies to bind LAP-TGEpi in ECM.
  • P3U 1 cells were incubated in round bottom tissue culture plates for 48 hours. Cells were then removed, leaving behind ECM on the surface of the plates. Three different groups were compared: (a) P3U1 cells expressing human LAP-TGFp 1 , (b) P3U1 cells expressing murine LAP-TGEP 1 , and (c) P3U1 cells without LAP- TGFp 1 (null cells). Binding of antibody to LAP-TGEP 1/ECM was then determined using biotinylated anti-LAP antibodies followed by incubation with streptavidin horseradish peroxidase (HRP) and 3',5,5'-tetramethylbenzidine (TMB) substrate.
  • HRP horseradish peroxidase
  • TMB 3',5,5'-tetramethylbenzidine
  • This Example describes the binding of anti-LAP antibodies to platelets and their effects on platelet degranulation.
  • a direct platelet binding assay was performed by flow cytometry. Diluted whole human blood was incubated with the indicated concentrations of directly conjugated anti-LAP antibodies (28Gl l_(hyb), 20E6_mIgG2a, 22F9_mIgG2a, l7G8_MgGl, and 24E3_MgGl) for 15 minutes. The reactions were then incubated for an additional 15 minutes with a commercially available directly conjugated antibody against CD61 (Bio Legend), and analyzed by flow cytometry. The data represents the anti-LAP mean fluorescence intensity of CD61 positive platelets. As shown in Figure 8, anti-LAP antibodies 28G11, 20E6, 22F9, 17G8, and 24E3 showed binding to platelets in a dose-responsive manner.
  • anti-LAP antibodies were further tested for platelet degranulation. Briefly, diluted whole human blood was incubated with the indicated concentrations of anti-LAP antibodies or adenosine diphosphate (ADP) as a positive control for 15 minutes. The reactions were then incubated for an additional 15 minutes with directly conjugated antibodies against CD61, to detect whole blood platelets, and CD62P (BioLegend) to detect degranulated platelets. The samples were analyzed by flow cytometry to determine the percentage of CD62P+ platelets.
  • ADP adenosine diphosphate
  • This Example describes the binding of anti-LAP antibodies to different types of immune cells.
  • THP-1 cells a cell line derived from a patient with acute monocytic leukemia that is reported to express LRRC33.
  • THP- 1 cells were incubated with FACS buffer and human Fc block followed by incubation with varying concentrations of Alexa 647 conjugated 28Gl l_(hyb), 22F9_mIgG2a, 20E6_mIgG2a, 17G8_MgGl, 24E3_MgGl, 2C9_mIgG2a, or mIgG2a isotype control.
  • THP-l cells were incubated with FACS buffer and human Fc block followed by incubation with 5 ug/ml of Alexa 647 conjugated 28G1 l_hyb, 22F9_mIgG2a, 20E6_mIgG2a, 2C9_mIgG2a, or IgG2a isotype control.
  • Cells were analyzed by flow cytometry and gated as single cells. Representative dot plots are shown in Figure 10D; in these plots, antibodies were at 5 ug/ml.
  • Anti-LAP antibodies were tested for their ability to bind to U937 cells, a myeloid cell line derived from a patient with histiocytic lymphoma.
  • U937 cells were incubated with FACS buffer and human Fc block followed by incubation with varying concentrations of Alexa 647 conjugated 28Gl l_hyb, 22F9_mIgG2a, or 20E6_mIgG2a.
  • Anti-LAP antibodies were tested for their ability to bind to immune cells isolated from mice carrying CT26 tumors. Briefly, lxlO 6 CT26 cells were injected into the flank region of 6 male Balb/C mice. When mean tumor volumes reached about 80 mm 3 , mice were treated with either IgG2a or IgGl isotype control antibodies at 10 mg/kg (this was originally part of an experiment in which mice were treated with therapeutic antibodies and these animals were intended to serve as controls). Mice were treated again 3 days later and harvested 7 days post first injection. Tumor tissue was disassociated in a GentlMACS dissociator and digested with Collagenase IV/ DNasel, strained through a 70-pm cell strainer and counted.
  • Spleen tissue was dissociated by passing through a 70-pm cell strainer and counted.
  • Cells were analyzed by flow cytometry using the following scheme: Gate on live cel ls-> Gate on single cells- Gate on CD45+ cells- Gate on CD41- population- ⁇ Gate on appropriate immune cell subsets as follows:
  • Binding of the anti-LAP antibodies was analyzed using Alexa 647-labeled 28Gl l-IgG2a, 22F9-IgG2a, and 20E6-IgG2a. A summary of the data is shown in Table 8 and in Figure 11.
  • the three anti-LAP antibodies 28G11, 22F9 and 20E6 display very different binding profiles to clinically relevant immune cell subsets in tumor tissue. Most notably, 22F9 and 20E6 bind to a higher percentage of regulatory T cells, M2 macrophages, and M-MDSCs compared to 28G11.
  • both 22F9 and 20E6 bind a higher percentage of regulatory T cells, M2 macrophages, and M-MDSC than does 28G11. This supports the superiority of 22F9 and 20E6 over 28G11 for the modulation of these important immunosuppressive cell populations in tumors.
  • the differences in binding of 22F9 and 20E6 support the potential preferential use of one or the other antibody in a given tumor depending on the makeup of the infiltrating leukocyte population.
  • CD 14+ cells were obtained from StemExpress where they were isolated from whole blood of a donor via magnetic negative selection.
  • Cells were cultured for 6 days in Immunocult macrophage medium (StemCell tech) + M-CSF (50 ng/ml) with the following additions to skew the cells to specific macrophage subtypes ( Ml macrophages: 50 ng/ml h-IFN-g + 10 ng/ml LPS; M2a macrophages: 10 ng/ml h-IL4; M2b macrophages: immobilized IgG + 100 ng/ml LPS; M2c macrophages: 10 ng/ml IL10 + 20 ng/ml TGFP). Cells were stained with CD 14 and Alexa 647-labeled anti-LAP antibodies
  • the anti-LAP antibodies displayed very different binding patterns to isolated human macrophage subsets. Notably, 22F9 bound a much higher percentage of all macrophage subpopulations than did 28G11 or 20E6. 20E6 bound a higher percentage of Ml macrophages than did 28G11.
  • CD4+ cells were isolated from PBMCs using magnetic negative selection according to instructions provided by the manufacturer (StemCell Tech). Cells were activated using a 1:1 ratio of Dynabeads (Thermo) to cells and cultured in advanced RPMI +10% FBS +30 U/ml human IL2 for 48 hours.
  • Cells were stained with live/dead dye followed by CD4, CD25, and 28G1 l-IgG2a, 22F9-IgG2a, 20E6-IgG2a, 17G8- hlgGl, 24E3-hIgGl, 2F8_(hyb), IgGl isotype control, or IgG2a isotype control for LAP expression.
  • Cells were fixed and permeabilized for Foxp3 staining according to the
  • Increased binding to specific cell populations is expected to be associated with direct clinical benefit.
  • the antibodies described here inhibit TGFP activation and release of the mature cytokine. Because TGFP acts in an autocrine or near-paracrine manner, selective binding to specific cell populations will result in inhibition of the production of mature TGFP in the immediate proximity of the indicated cell population.
  • the increased binding of anti-LAP antibody 20E6 when compared to antibody 28G11 to regulatory T cells would be expected to result in selectively reduced TGFP levels at the surface of those same regulatory T cells.
  • TGFP is a major driver of regulatory T cell generation, this is expected to result in reduced numbers of regulatory T cells in the tumor microenvironment and increased clinical efficacy of 20E6 over 28G11.
  • the increased binding of anti-LAP antibody 22F9 when compared to antibody 28G11 to macrophage subsets would be expected to result in selectively reduced TGFP levels at the surface of those same macrophages.
  • TGFP is a primary mechanism of cell-contact dependent macrophage inhibition of effector T cell function, this is expected to result in reduced macrophage-mediated inhibition and increased effector T cell function in the tumor microenvironment and increased clinical efficacy of 20E6 over 28G11.
  • the anti-LAP antibodies are of an isotype with active effector function and enhanced binding of a specific anti-LAP antibody to a given cell population will result in increased depletion of that cell population by ADCC or CDC.
  • ADCC or CDC the increased binding of anti-LAP antibody 20E6 when compared to antibody 28G11 to regulatory T cells would be expected to result in increased ADCC or CDC-mediated depletion of those regulatory T cells in the tumor microenvironment and increased clinical efficacy of 20E6 over 28G11.
  • the increased binding of anti-LAP antibody 22F9 when compared to antibody 28G11 to macrophage subsets would be expected to result in increased ADCC- or CDC-mediated depletion of those macrophage subsets in the tumor microenvironment and increased clinical efficacy of 22F9 over 28G11.
  • the data presented in this example demonstrate that the finding that the anti-LAP antibodies described here bind differently to immune cell subpopulations can be demonstrated in both murine and human systems and in both primary cell populations and transformed cell lines.

Abstract

L'invention concerne des anticorps anti-LAP (par exemple, des anticorps anti-LAP humanisés, chimériques et humains recombinants) ou des fragments de liaison à l'antigène de ceux-ci qui ont des propriétés thérapeutiquement bénéfiques, telles que la liaison spécifiquement à ΡΑΡ-ΤΟΡβ1 sur des cellules mais pas à LAP-TGFβ1 dans la matrice extracellulaire, ainsi que des compositions les comprenant. L'invention concerne également des utilisations de ces anticorps ou fragments de liaison à l'antigène dans des applications thérapeutiques, telles que dans le traitement du cancer, et des applications diagnostiques.
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Family Cites Families (201)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4444887A (en) 1979-12-10 1984-04-24 Sloan-Kettering Institute Process for making human antibody producing B-lymphocytes
US4475196A (en) 1981-03-06 1984-10-02 Zor Clair G Instrument for locating faults in aircraft passenger reading light and attendant call control system
US4447233A (en) 1981-04-10 1984-05-08 Parker-Hannifin Corporation Medication infusion pump
US4439196A (en) 1982-03-18 1984-03-27 Merck & Co., Inc. Osmotic drug delivery system
US4522811A (en) 1982-07-08 1985-06-11 Syntex (U.S.A.) Inc. Serial injection of muramyldipeptides and liposomes enhances the anti-infective activity of muramyldipeptides
US4716111A (en) 1982-08-11 1987-12-29 Trustees Of Boston University Process for producing human antibodies
US4447224A (en) 1982-09-20 1984-05-08 Infusaid Corporation Variable flow implantable infusion apparatus
US4487603A (en) 1982-11-26 1984-12-11 Cordis Corporation Implantable microinfusion pump system
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4486194A (en) 1983-06-08 1984-12-04 James Ferrara Therapeutic device for administering medicaments through the skin
EP0154316B1 (fr) 1984-03-06 1989-09-13 Takeda Chemical Industries, Ltd. Lymphokine chimiquement modifiée et son procédé de préparation
US4596556A (en) 1985-03-25 1986-06-24 Bioject, Inc. Hypodermic injection apparatus
DE3590766C2 (fr) 1985-03-30 1991-01-10 Marc Genf/Geneve Ch Ballivet
US5374548A (en) 1986-05-02 1994-12-20 Genentech, Inc. Methods and compositions for the attachment of proteins to liposomes using a glycophospholipid anchor
MX9203291A (es) 1985-06-26 1992-08-01 Liposome Co Inc Metodo para acoplamiento de liposomas.
US5618920A (en) 1985-11-01 1997-04-08 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
DE3600905A1 (de) 1986-01-15 1987-07-16 Ant Nachrichtentech Verfahren zum dekodieren von binaersignalen sowie viterbi-dekoder und anwendungen
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
GB8607679D0 (en) 1986-03-27 1986-04-30 Winter G P Recombinant dna product
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US5260203A (en) 1986-09-02 1993-11-09 Enzon, Inc. Single polypeptide chain binding molecules
US4881175A (en) 1986-09-02 1989-11-14 Genex Corporation Computer based system and method for determining and displaying possible chemical structures for converting double- or multiple-chain polypeptides to single-chain polypeptides
US5567610A (en) 1986-09-04 1996-10-22 Bioinvent International Ab Method of producing human monoclonal antibodies and kit therefor
US5763192A (en) 1986-11-20 1998-06-09 Ixsys, Incorporated Process for obtaining DNA, RNA, peptides, polypeptides, or protein, by recombinant DNA technique
DE3883899T3 (de) 1987-03-18 1999-04-22 Sb2 Inc Geänderte antikörper.
US5013653A (en) 1987-03-20 1991-05-07 Creative Biomolecules, Inc. Product and process for introduction of a hinge region into a fusion protein to facilitate cleavage
US5132405A (en) 1987-05-21 1992-07-21 Creative Biomolecules, Inc. Biosynthetic antibody binding sites
US5258498A (en) 1987-05-21 1993-11-02 Creative Biomolecules, Inc. Polypeptide linkers for production of biosynthetic proteins
US5091513A (en) 1987-05-21 1992-02-25 Creative Biomolecules, Inc. Biosynthetic antibody binding sites
JPH02500329A (ja) 1987-05-21 1990-02-08 クリエイテイブ・バイオマリキユールズ・インコーポレーテツド ターゲット化多機能蛋白質
US4790824A (en) 1987-06-19 1988-12-13 Bioject, Inc. Non-invasive hypodermic injection device
US4941880A (en) 1987-06-19 1990-07-17 Bioject, Inc. Pre-filled ampule and non-invasive hypodermic injection device assembly
US5677425A (en) 1987-09-04 1997-10-14 Celltech Therapeutics Limited Recombinant antibody
CA2006596C (fr) 1988-12-22 2000-09-05 Rika Ishikawa G-csf modifie chimiquement
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5108921A (en) 1989-04-03 1992-04-28 Purdue Research Foundation Method for enhanced transmembrane transport of exogenous molecules
CA2016842A1 (fr) 1989-05-16 1990-11-16 Richard A. Lerner Methode pour puiser dans le repertoire immunologique
CA2016841C (fr) 1989-05-16 1999-09-21 William D. Huse Methode de production de polymeres ayant une activite choisie
EP0478627A4 (en) 1989-05-16 1992-08-19 William D. Huse Co-expression of heteromeric receptors
DE3920358A1 (de) 1989-06-22 1991-01-17 Behringwerke Ag Bispezifische und oligospezifische, mono- und oligovalente antikoerperkonstrukte, ihre herstellung und verwendung
US5312335A (en) 1989-11-09 1994-05-17 Bioject Inc. Needleless hypodermic injection device
US5064413A (en) 1989-11-09 1991-11-12 Bioject, Inc. Needleless hypodermic injection device
GB8928874D0 (en) 1989-12-21 1990-02-28 Celltech Ltd Humanised antibodies
CA2050918A1 (fr) 1990-01-12 1991-07-13 Raju Kucherlapati Production d'anticorps xenogeniques
US5229275A (en) 1990-04-26 1993-07-20 Akzo N.V. In-vitro method for producing antigen-specific human monoclonal antibodies
GB9015198D0 (en) 1990-07-10 1990-08-29 Brien Caroline J O Binding substance
EP0544809B1 (fr) 1990-08-24 1998-12-16 Ixsys, Inc. Procede de production par synthese d'oligonucleotides ayant des codons aleatoires
EP0563296B1 (fr) 1990-12-20 1999-03-17 Ixsys, Inc. Optimalisation de proteines de liaison
EP0519596B1 (fr) 1991-05-17 2005-02-23 Merck & Co. Inc. Procédé pour réduire l'immunogénicité des domaines variables d'anticorps
DE69229477T2 (de) 1991-09-23 1999-12-09 Cambridge Antibody Tech Methoden zur Herstellung humanisierter Antikörper
ES2136092T3 (es) 1991-09-23 1999-11-16 Medical Res Council Procedimientos para la produccion de anticuerpos humanizados.
AU3178993A (en) 1991-11-25 1993-06-28 Enzon, Inc. Multivalent antigen-binding proteins
CA2103887C (fr) 1991-12-13 2005-08-30 Gary M. Studnicka Methodes et materiaux pour la preparation de domaines variables d'anticorps modifies et leurs utilisations therapeutiques
US5714350A (en) 1992-03-09 1998-02-03 Protein Design Labs, Inc. Increasing antibody affinity by altering glycosylation in the immunoglobulin variable region
EP0640094A1 (fr) 1992-04-24 1995-03-01 The Board Of Regents, The University Of Texas System Production recombinante de domaines semblables a l'immunoglobuline dans des cellules procaryotes
US5383851A (en) 1992-07-24 1995-01-24 Bioject Inc. Needleless hypodermic injection device
DK0656946T4 (da) 1992-08-21 2010-07-26 Univ Bruxelles Immunoglobuliner uden lette kæder
US6005079A (en) 1992-08-21 1999-12-21 Vrije Universiteit Brussels Immunoglobulins devoid of light chains
US5639641A (en) 1992-09-09 1997-06-17 Immunogen Inc. Resurfacing of rodent antibodies
DK0669836T3 (da) 1992-11-13 1996-10-14 Idec Pharma Corp Terapeutisk anvendelse af kimære og radioaktivt mærkede antistoffer og humant B-lymfocytbegrænset differentieringsantigen til behandling af B-cellelymfom
AU6132994A (en) 1993-02-02 1994-08-29 Scripps Research Institute, The Methods for producing antibody libraries using universal or randomized immunoglobulin light chains
WO1994025591A1 (fr) 1993-04-29 1994-11-10 Unilever N.V. PRODUCTION D'ANTICORPS OU DE FRAGMENTS FONCTIONNALISES D'ANTICORPS, DERIVES DES IMMUNOGLOBULINES A CHAINE LOURDE DE $i(CAMELIDAE)
EP0714409A1 (fr) 1993-06-16 1996-06-05 Celltech Therapeutics Limited Anticorps
JPH08507549A (ja) 1993-12-27 1996-08-13 バクスター、インターナショナル、インコーポレイテッド 水溶性の非免疫原性ポリアミド架橋剤
US6121022A (en) 1995-04-14 2000-09-19 Genentech, Inc. Altered polypeptides with increased half-life
US5869046A (en) 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
US6096871A (en) 1995-04-14 2000-08-01 Genentech, Inc. Polypeptides altered to contain an epitope from the Fc region of an IgG molecule for increased half-life
KR100654645B1 (ko) 1995-04-27 2007-04-04 아브게닉스, 인크. 면역화된 제노마우스 유래의 인간 항체
EP0823941A4 (fr) 1995-04-28 2001-09-19 Abgenix Inc Anticorps humains derives de xeno-souris immunisees
ATE483733T1 (de) 1995-06-14 2010-10-15 Univ California Hochaffine humane antikörper gegen tumorantigene
US6331431B1 (en) 1995-11-28 2001-12-18 Ixsys, Inc. Vacuum device and method for isolating periplasmic fraction from cells
AU728657B2 (en) 1996-03-18 2001-01-18 Board Of Regents, The University Of Texas System Immunoglobulin-like domains with increased half-lives
US5714352A (en) 1996-03-20 1998-02-03 Xenotech Incorporated Directed switch-mediated DNA recombination
US5916771A (en) 1996-10-11 1999-06-29 Abgenix, Inc. Production of a multimeric protein by cell fusion method
ATE549918T1 (de) 1996-12-03 2012-04-15 Amgen Fremont Inc Menschliche antikörper, die ausdrücklich menschliches tnf alpha binden
US6277375B1 (en) 1997-03-03 2001-08-21 Board Of Regents, The University Of Texas System Immunoglobulin-like domains with increased half-lives
JP2001523958A (ja) 1997-03-21 2001-11-27 ブライハム アンド ウィミンズ ホスピタル，インコーポレイテッド 免疫療法のｃｔｌａ−４結合ペプチド
EP0970126B1 (fr) 1997-04-14 2001-04-18 Micromet AG Nouveau procede de production de recepteurs d'anti-antigenes humains et leur utilisation
US6235883B1 (en) 1997-05-05 2001-05-22 Abgenix, Inc. Human monoclonal antibodies to epidermal growth factor receptor
ATE319745T1 (de) 1997-05-21 2006-03-15 Biovation Ltd Verfahren zur herstellung von nicht-immunogenen proteinen
US6891082B2 (en) 1997-08-01 2005-05-10 The Johns Hopkins University School Of Medicine Transgenic non-human animals expressing a truncated activintype II receptor
AU8691398A (en) 1997-08-04 1999-02-22 Ixsys, Incorporated Methods for identifying ligand specific binding molecules
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
ES2246567T3 (es) 1998-04-15 2006-02-16 Brigham & Womens Hospital Composiciones para receptores inhibidores de celulas t y usos de las mismas.
DE69942021D1 (de) 1998-04-20 2010-04-01 Glycart Biotechnology Ag Glykosylierungs-engineering von antikörpern zur verbesserung der antikörperabhängigen zellvermittelten zytotoxizität
IT1303776B1 (it) 1998-11-19 2001-02-23 S I S S A Scuola Internaz Supe Processo per la preparazione di genoteche, di polipeptidi utilizzandodette genoteche e i polipepti ottenuti.
CN1328571B (zh) 1998-12-23 2016-08-31 辉瑞大药厂 抗ctla-4的人单克隆抗体
EP2386574A3 (fr) 1999-01-15 2012-06-27 Genentech, Inc. Variantes de polypeptide et fonction effectrice altérée
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
DK2270147T4 (da) 1999-04-09 2020-08-31 Kyowa Kirin Co Ltd Fremgangsmåde til at kontrollere aktiviteten af immunologisk funktionelt molekyle
WO2001014556A1 (fr) 1999-08-23 2001-03-01 Dana-Farber Cancer Institute, Inc. Nouvelles molecules b7-4 et leurs utilisations
AU7950400A (en) 1999-10-19 2001-04-30 Kyowa Hakko Kogyo Co. Ltd. Process for producing polypeptide
WO2001039722A2 (fr) 1999-11-30 2001-06-07 Mayo Foundation For Medical Education And Research Nouvelle molecule immunoregulatrice b7-h1,
HUP0204475A2 (en) 2000-02-11 2003-04-28 Merck Patent Gmbh Enhancing the circulating half-life of antibody-based fusion proteins
DK1297172T3 (da) 2000-06-28 2006-02-13 Glycofi Inc Fremgangsmåder til frembringelse af modificerede glucoproteiner
US7449308B2 (en) 2000-06-28 2008-11-11 Glycofi, Inc. Combinatorial DNA library for producing modified N-glycans in lower eukaryotes
US6725230B2 (en) 2000-07-18 2004-04-20 Aegis Analytical Corporation System, method and computer program for assembling process data of multi-database origins using a hierarchical display
US6946292B2 (en) 2000-10-06 2005-09-20 Kyowa Hakko Kogyo Co., Ltd. Cells producing antibody compositions with increased antibody dependent cytotoxic activity
US6374470B1 (en) 2000-10-06 2002-04-23 Milliken & Company Face plate for spun-like textured yarn
US6989452B2 (en) 2001-05-31 2006-01-24 Medarex, Inc. Disulfide prodrugs and linkers and stabilizers useful therefor
US6884619B2 (en) 2001-07-17 2005-04-26 Yale University Inhibition of BEHAB cleavage and primary central nervous system (CNS) tumors
EP1423510A4 (fr) 2001-08-03 2005-06-01 Glycart Biotechnology Ag Variants de glycosylation d'anticorps presentant une cytotoxicite cellulaire accrue dependante des anticorps
KR100988949B1 (ko) 2001-10-25 2010-10-20 제넨테크, 인크. 당단백질 조성물
US20040002587A1 (en) 2002-02-20 2004-01-01 Watkins Jeffry D. Fc region variants
US20040132101A1 (en) 2002-09-27 2004-07-08 Xencor Optimized Fc variants and methods for their generation
US7317091B2 (en) 2002-03-01 2008-01-08 Xencor, Inc. Optimized Fc variants
WO2003086310A2 (fr) 2002-04-12 2003-10-23 Ramot At Tel Aviv University Ltd. Prevention de l'inflammation du cerveau en tant que resultat d'une reponse auto-immune induite
WO2005117986A2 (fr) 2004-06-01 2005-12-15 Genentech, Inc. Conjugues de medicaments anticorps et procedes correspondants
IL149820A0 (en) 2002-05-23 2002-11-10 Curetech Ltd Humanized immunomodulatory monoclonal antibodies for the treatment of neoplastic disease or immunodeficiency
US7425620B2 (en) 2002-08-14 2008-09-16 Scott Koenig FcγRIIB-specific antibodies and methods of use thereof
DK1553975T3 (da) 2002-09-27 2012-05-07 Xencor Inc Optimerede Fc-varianter og fremgangsmåder til generering heraf.
PT1562972E (pt) 2002-10-15 2010-11-10 Facet Biotech Corp Alteração de afinidades de ligação ao fcrn ou semi-vidas séricas de anticorpos por mutagénese
EP2368578A1 (fr) 2003-01-09 2011-09-28 Macrogenics, Inc. Identification et ingénierie d'anticorps avec régions FC de variante et procédés d'utilisation associés
US7410483B2 (en) 2003-05-23 2008-08-12 Novare Surgical Systems, Inc. Hand-actuated device for remote manipulation of a grasping tool
WO2005013915A2 (fr) 2003-08-08 2005-02-17 The Regents Of The University Of California Nouvelles indications permettant de transformer les regulateurs du facteur de croissance beta
WO2005023870A1 (fr) 2003-09-04 2005-03-17 Riken Anticorps capable de reconnaitre la region de controle de l'activation de tgf-$g(b)
US8101720B2 (en) 2004-10-21 2012-01-24 Xencor, Inc. Immunoglobulin insertions, deletions and substitutions
GB0324368D0 (en) 2003-10-17 2003-11-19 Univ Cambridge Tech Polypeptides including modified constant regions
BRPI0506771A (pt) 2004-01-12 2007-05-22 Applied Molecular Evolution anticorpo, e, composição farmacêutica
AU2005227326B2 (en) 2004-03-24 2009-12-03 Xencor, Inc. Immunoglobulin variants outside the Fc region
RU2386638C2 (ru) 2004-03-31 2010-04-20 Дженентек, Инк. Гуманизированные анти-тфр-бета-антитела
WO2005117973A2 (fr) 2004-05-05 2005-12-15 Merrimack Pharmaceuticals, Inc. Agents de liaison bispecifiques pour la modulation de l'activite biologique
EP1747021B1 (fr) 2004-05-19 2015-09-09 E. R. Squibb & Sons, L.L.C. Bras de liaison chimiques et conjugues associes
US7691962B2 (en) 2004-05-19 2010-04-06 Medarex, Inc. Chemical linkers and conjugates thereof
EP1766396B1 (fr) 2004-06-07 2010-08-11 Ramot at Tel-Aviv University Ltd. Procede d'immunisation passive contre des maladies ou des troubles caracterise(e)s par agregation amyloide a risque diminue de neuroinflammation
JP2008512353A (ja) 2004-07-21 2008-04-24 グライコフィ， インコーポレイテッド 主にＧｌｃＮＡｃ２Ｍａｎ３ＧｌｃＮＡｃ２グリコフォームを含むイムノグロブリン
ES2426816T3 (es) 2004-08-04 2013-10-25 Mentrik Biotech, Llc Regiones Fc variantes
MX2007006117A (es) 2004-11-23 2007-07-13 Pip Co Ltd Caja de llave de servicio de agua empotrada en la pared.
CN101163502A (zh) 2005-02-08 2008-04-16 根茨美公司 针对TGFβ的抗体
JP2008531557A (ja) 2005-02-23 2008-08-14 メリマック ファーマシューティカルズ インコーポレーティッド 生物活性を調節するための二重特異性結合剤
US7714016B2 (en) 2005-04-08 2010-05-11 Medarex, Inc. Cytotoxic compounds and conjugates with cleavable substrates
BRPI0608376A8 (pt) 2005-04-22 2018-10-16 Lilly Co Eli composição de ligação, método de uso de uma composição de ligação, e, kit de detecção
EA026785B1 (ru) 2005-05-10 2017-05-31 Инсайт Холдингс Корпорейшн Модуляторы индоламин 2,3-диоксигеназы и способы их применения
AU2006294554B2 (en) 2005-09-26 2013-03-21 E. R. Squibb & Sons, L.L.C. Antibody-drug conjugates and methods of use
SI1940789T1 (sl) 2005-10-26 2012-03-30 Medarex Inc Postopki in spojine za pripravo cc analogov
CA2627190A1 (fr) 2005-11-10 2007-05-24 Medarex, Inc. Composes et conjugues cytotoxiques
ES2540561T3 (es) 2005-12-20 2015-07-10 Incyte Corporation N-hidroxiamidinoheterociclos como moduladores de indolamina 2,3-dioxigenasa
US20080125470A1 (en) 2006-09-19 2008-05-29 Incyte Corporation N-hydroxyamidinoheterocycles as modulators of indoleamine 2,3-dioxygenase
CL2007002650A1 (es) 2006-09-19 2008-02-08 Incyte Corp Compuestos derivados de heterociclo n-hidroxiamino; composicion farmaceutica, util para tratar cancer, infecciones virales y desordenes neurodegenerativos entre otras.
US20080227704A1 (en) 2006-12-21 2008-09-18 Kamens Joanne S CXCL13 binding proteins
TWI412367B (zh) 2006-12-28 2013-10-21 Medarex Llc 化學鏈接劑與可裂解基質以及其之綴合物
CL2008000510A1 (es) 2007-02-21 2008-08-22 Medarex Inc Compuestos conjugados farmaco-ligandos, que se unen a citotoxinas potentes; composicion farmaceutica; y uso para retardar o detener el crecimiento de un tumor en un mamifero.
US7732401B2 (en) 2007-03-08 2010-06-08 Riken Inhibitor of TGF-β activation reaction
EP1987839A1 (fr) 2007-04-30 2008-11-05 I.N.S.E.R.M. Institut National de la Sante et de la Recherche Medicale Anticorps monoclonal cytotoxique anti-LAG-3 et son utilisation pour le traitement ou la prévention d'un rejet de greffe d'organe et de maladies auto-immunes
US9244059B2 (en) 2007-04-30 2016-01-26 Immutep Parc Club Orsay Cytotoxic anti-LAG-3 monoclonal antibody and its use in the treatment or prevention of organ transplant rejection and autoimmune disease
US20090028857A1 (en) 2007-07-23 2009-01-29 Cell Genesys, Inc. Pd-1 antibodies in combination with a cytokine-secreting cell and methods of use thereof
EP2044949A1 (fr) 2007-10-05 2009-04-08 Immutep Utilisation de lag-3 recombinant ou ses dérivatifs pour déclencher la réponse immune des monocytes
US20090181037A1 (en) 2007-11-02 2009-07-16 George Heavner Semi-Synthetic GLP-1 Peptide-FC Fusion Constructs, Methods and Uses
CA2707308C (fr) 2007-11-30 2016-08-02 Newlink Genetics Corporation Inhibiteurs de l'ido
JP5470817B2 (ja) 2008-03-10 2014-04-16 日産自動車株式会社 電池用電極およびこれを用いた電池、並びにその製造方法
WO2009114335A2 (fr) 2008-03-12 2009-09-17 Merck & Co., Inc. Protéines de liaison avec pd-1
US8674079B2 (en) 2008-03-27 2014-03-18 Tohoku University Cancer cell migration and cancer cell invasion inhibitor
AR072999A1 (es) 2008-08-11 2010-10-06 Medarex Inc Anticuerpos humanos que se unen al gen 3 de activacion linfocitaria (lag-3) y los usos de estos
SG196798A1 (en) 2008-12-09 2014-02-13 Genentech Inc Anti-pd-l1 antibodies and their use to enhance t-cell function
EP2421559B1 (fr) 2009-04-24 2016-07-20 Vanderbilt University Induction anti-tgf-beta de la fonction de la croissance osseuse
US20110007023A1 (en) 2009-07-09 2011-01-13 Sony Ericsson Mobile Communications Ab Display device, touch screen device comprising the display device, mobile device and method for sensing a force on a display device
IN2012DN01663A (fr) 2009-09-16 2015-06-05 Immunomedics Inc
ES2601226T3 (es) 2009-10-28 2017-02-14 Newlink Genetics Corporation Derivados de imidazol como inhibidores de IDO
CA2778714C (fr) 2009-11-24 2018-02-27 Medimmune Limited Agents de liaison cibles diriges contre b7-h1
CN102791738B (zh) 2009-12-10 2015-10-07 霍夫曼-拉罗奇有限公司 优先结合人csf1r胞外域4的抗体及其用途
WO2011102483A1 (fr) 2010-02-19 2011-08-25 独立行政法人理化学研究所 ANTICORPS SE LIANT AU TGF-β DU LAP HUMAIN
PL2542256T3 (pl) 2010-03-04 2020-01-31 Macrogenics, Inc. Przeciwciała reaktywne wobec b7-h3, ich immunologicznie czynne fragmenty i ich zastosowania
EP2542588A1 (fr) 2010-03-05 2013-01-09 F. Hoffmann-La Roche AG Anticorps contre le csf-1r humain et leurs utilisations
EP2542587A1 (fr) 2010-03-05 2013-01-09 F. Hoffmann-La Roche AG Anticorps dirigés contre le csf-1r humain et utilisations associées
EP3943154A1 (fr) 2010-05-04 2022-01-26 Five Prime Therapeutics, Inc. Anticorps se liant au csf1r
US8907053B2 (en) 2010-06-25 2014-12-09 Aurigene Discovery Technologies Limited Immunosuppression modulating compounds
CN103119066B (zh) 2010-08-30 2016-04-20 独立行政法人理化学研究所 具有抑制TGF-β受体活化的活性的化合物、该化合物的筛选方法、以及用于预防或治疗由丙型肝炎病毒引起的疾病的组合物
SG10201506906VA (en) 2010-09-09 2015-10-29 Pfizer 4-1bb binding molecules
NO2694640T3 (fr) 2011-04-15 2018-03-17
ES2669310T3 (es) 2011-04-20 2018-05-24 Medimmune, Llc Anticuerpos y otras moléculas que se unen con B7-H1 y PD-1
TW201311895A (zh) 2011-07-27 2013-03-16 Baylor Res Inst 用於胰臟癌之樹突細胞（ｄｃ）－疫苗療法
AU2013201121A1 (en) 2011-09-20 2013-04-04 Vical Incorporated Synergistic anti-tumor efficacy using alloantigen combination immunotherapy
US9782452B2 (en) 2011-11-22 2017-10-10 Cornell University Methods for stimulating hematopoietic recovery by inhibiting TGFβ signaling
PT2785375T (pt) 2011-11-28 2020-10-29 Merck Patent Gmbh Anticorpos anti-pd-l1 e usos destes
JP6242804B2 (ja) 2011-12-15 2017-12-06 エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト ヒトｃｓｆ−１ｒに対する抗体及びその使用
MX2014008961A (es) 2012-02-06 2014-10-14 Genentech Inc Composiciones y metodos para utilizar inhibidores de csf1r.
US9518112B2 (en) 2012-03-08 2016-12-13 Ludwig Institute For Cancer Research Ltd TGF-β1 specific antibodies and methods and uses thereof
AR090263A1 (es) 2012-03-08 2014-10-29 Hoffmann La Roche Terapia combinada de anticuerpos contra el csf-1r humano y las utilizaciones de la misma
KR20220162819A (ko) 2012-05-11 2022-12-08 파이브 프라임 테라퓨틱스, 인크. 콜로니 자극 인자 1 수용체(csf1r)에 결속하는 항체들에 의한 질병 상태의 치료 방법
SG10201700698WA (en) 2012-05-15 2017-02-27 Bristol Myers Squibb Co Cancer immunotherapy by disrupting pd-1/pd-l1 signaling
AR091649A1 (es) 2012-07-02 2015-02-18 Bristol Myers Squibb Co Optimizacion de anticuerpos que se fijan al gen de activacion de linfocitos 3 (lag-3) y sus usos
CN104684582A (zh) 2012-08-31 2015-06-03 戊瑞治疗有限公司 用结合群落刺激因子1受体(csf1r)的抗体治疗病状的方法
CA3201072A1 (fr) 2012-10-12 2014-04-17 The Brigham And Women's Hospital, Inc. Renforcement de la reponse immunitaire
SG11201503271XA (en) 2012-11-06 2015-05-28 Scholar Rock Inc Compositions and methods for modulating cell signaling
JP6136279B2 (ja) 2013-01-15 2017-05-31 株式会社ジェイテクト 転がり軸受装置
TWI503850B (zh) 2013-03-22 2015-10-11 Polytronics Technology Corp 過電流保護元件
CA2911514A1 (fr) 2013-05-06 2014-11-13 Scholar Rock, Inc. Compositions et procedes de modulation du facteur de croissance
US9574476B2 (en) 2013-08-01 2017-02-21 Elwha Llc Systems, methods, and apparatuses related to vehicles with reduced emissions
TWI510996B (zh) 2013-10-03 2015-12-01 Acer Inc 控制觸控面板的方法以及使用該方法的可攜式電腦
EP2878308B1 (fr) 2013-12-02 2018-10-31 Thomas Harder Agents et procédés pour la suppression de l'activation des lymphocytes T
CA2947967A1 (fr) 2014-05-06 2015-11-12 Scholar Rock, Inc. Compositions et procedes de modulation de facteur de croissance
JP7152156B2 (ja) * 2015-01-14 2022-10-12 ザ・ブリガーム・アンド・ウーメンズ・ホスピタル・インコーポレーテッド 抗ｌａｐモノクローナル抗体による癌の処置
ES2820768T3 (es) 2015-04-03 2021-04-22 Xoma Technology Ltd Tratamiento del cáncer usando inhibidores de TGF-beta y PD-1
US11643459B2 (en) 2016-03-11 2023-05-09 Scholar Rock, Inc. TGFβ1-binding immunoglobulins and use thereof
WO2018013939A1 (fr) 2016-07-14 2018-01-18 Scholar Rock, Inc. Anticorps anti-tgfb, méthodes et utilisations
WO2018043734A1 (fr) 2016-09-05 2018-03-08 Chugai Seiyaku Kabushiki Kaisha Anticorps anti-tgf-beta 1 et leurs procédés d'utilisation
US9816280B1 (en) 2016-11-02 2017-11-14 Matthew Reitnauer Portable floor
CA3049005A1 (fr) 2017-01-06 2018-07-12 Scholar Rock, Inc. Inhibiteurs specifiques d'une isoforme, permissifs au contexte de tgf.beta.1 et leur utilisation
EP3621694B1 (fr) 2017-05-09 2023-07-05 Scholar Rock, Inc. Inhibiteurs de lrrc33 et utilisations de ceux-ci
EP3658583A1 (fr) 2017-07-28 2020-06-03 Scholar Rock, Inc. Inhibiteurs spécifiques du complexe ltbp de tgf-bêta 1 et leurs utilisations
US11230601B2 (en) 2017-10-10 2022-01-25 Tilos Therapeutics, Inc. Methods of using anti-lap antibodies

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US20220017612A1 (en) 2022-01-20
TW202035445A (zh) 2020-10-01
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US20200140530A1 (en) 2020-05-07

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